Linepithema humile) for RNA viruses. We used metatranscriptomic analysis, which identified six potential novel viruses in the Dicistroviridae family. Of these, three contigs were confirmed by Sanger sequencing as Linepithema humile virus-1 (LHUV-1), a novel strain of Kashmir bee virus (KBV) and Black queen cell virus (BQCV), while the others were chimeric or misassembled sequences. We extended the known sequence of LHUV-1 to confirm its placement in the Dicistroviridae and categorised its relationship to closest relatives, which were all viruses infecting Hymenoptera. We examined further for known viruses by mapping our metatranscriptomic sequences to all viral genomes, and confirmed KBV, BQCV, LHUV-1 and Deformed wing virus (DWV) presence using qRT-PCR. Viral replication was confirmed for DWV, KBV and LHUV-1. Viral titers in ants were higher in the presence of honey bee hives. Argentine ants appear to host a range of’ honey bee’ pathogens in addition to a virus currently described only from this invasive ant. The role of these viruses in the population dynamics of the ant remain to be determined, but offer potential targets for biocontrol approaches.Social insects host a diversity of viruses. We examined New Zealand populations of the globally widely distributed invasive Argentine ant ( A recent review noted honey bees host 24 viruses, primarily in the Dicistroviridae and Iflaviridae families3. Of these, the Deformed wing virus (DWV) has been suggested as a likely candidate for the majority of global honey bee colony losses during the past 50 years4. Such viruses, however, are not restricted to honey bees. There is increasing evidence that these ‘honey bee’ viruses are common in a wide range of insect hosts7.Social insects carry a range of viruses that can have a major effect on host population dynamics. Perhaps the best known viral community is from honey bees, which has been the focus of considerable study due to their economic importance. A range of different factors are likely to contribute to colony collapse and bee declines in general, with viruses frequently considered key playersSolenopsis invicta)8. These were the first viruses fully described from ants. Three of these viruses are positive-sense, single-stranded RNA (ssRNA) viruses, with one  assigned taxonomically to the Dicistroviridae family, one  in a proposed new family, Solinviviridae9 and the third currently unclassified 9; The fourth virus is a DNA virus, and has been placed in the family Parvoviridae10. One of the three ssRNA viruses, SINV-3, shows promise as a biocontrol agent as it can cause significant mortality in laboratory fire ant colonies11. Metatranscriptomic and pyrosequencing techniques have proven particularly useful for viral discovery in these and other ant species . Using these and complementary approaches we have found the invasive Argentine ant (Linepithema humile) to host a previously undescribed virus Linepithema humile virus-1 (LHUV-1) and DWV7. Replication of both viruses was confirmed within Argentine ants indicating that these viruses were parasitizing their hosts. Our previous work recovered 1,200 nucleotides of the LHUV-1 virus, but did not recover the entire non-structural polyprotein region which would have confirmed accurate phylogenetic placement. In that work we also noted evidence for the likely presence of other novel and known viruses. We found other novel contigs of viral origin , but were unable to characterise these further.Other social insects have been found to carry their own unique suites of viral pathogens. For example, over the last decade four viruses have been described from the red imported fire ant , Black queen cell virus (BQCV), and Acute bee paralysis virus (ABPV), as well as other ssRNA viruses known to affect insects, and two dsDNA viruses , BQCV or Drosophila C virus , and was identical to the Helicase protein for BQCV (GenBank accession ABS82427). For TR30069|c0 1519 base pairs were recovered, which, matched most closely to KBV, IAPV, ABPV and Formica exsecta 1 virus (FEX-1) non-structural polyprotein. HHpred predicted an ORF of 313 codons to partially include the RNA dependent RNA polymerase (RdRp) protein  most closely matching KBV. As we could not recover the entire RdRp protein for TR30069|c0 we do not present further results for this contig. TR30069|c1 was recovered in 3 fragments . GenBank blastp searches matched all fragments most closely to Israeli acute paralysis virus (IAPV), KBV, FEX-1 and ABPV. HHpred did not predict functional viral proteins in the longest fragment. The second fragment contained an ORF of 100 codons that HHpred predicted to include part of Protease 3CG . HHpred predicted the third fragment with an ORF of 252 codons to partially span RdRp . The TR30069|c1 RdRp fragment matched LHUV-1 RdRp, and the Protease 3CG matched contig n1905. We concluded the TR30069|c1 contig was chimeric owing to misassembly, and thus excluded it from further analysis.We identified six putative viral contigs using Blast2GO23 to computationally extend the sequence information for LHUV-1 from 1,200 to 8,268 bases, and designed primers for confirmatory Sanger sequencing (Supplementary methods). Sanger sequencing recovered 7,239 bases. None of the newly identified contigs  or previously identified contigs  aligned with the extended LHUV-1 sequence. The LHUV-1 sequence translated into a single large ORF  of Dicistroviridae . The ORF2 sequence included the partial sequence of the capsid gene Dicistroviridae, the first ORF of n1905 encodes the non-structural polyproteins , and matched the KBV non-structural polyproteins on GenBank with 100% coverage and 99% identity. In Dicistroviridae the second ORF codes for the structural polyproteins. Our second ORF coded for capsid polyproteins, and matched KBV ORFs on GenBank with 100% coverage and 98% identity.We extended the n1905 contig to 5,840 bases using IVA, which was further extended to 9,450 bases by Sanger sequencing. The resulting sequence matched closely to the two KBV genomes on GenBank (100% coverage and 92–96% identity). The n1905 contig contained a one large (1683 amino acids) and one smaller (843 amino acids) ORF. Like other Dicistroviridae viruses have genomes ranging from 8.5–10.2 kb. Thus we likely recovered at least 70% of the LHUV-1 genome, including the complete ORF1 region  and part of ORF2. The TR31304, TR30069|c0, TTR30069|c1, n1000 and n1050 contigs failed to extend using IVA, possibly because these were chimeric or fragmented assemblies.Poly(A) tails were not recovered for either LHUV-1 or n1905 extended sequences, indicating that we did not fully recover the genomes of either virus. Based on the published KBV genomes we recovered all but 37 5′ bases and 25 3′ bases (excluding the poly(A) tail). As LHUV-1 is a novel virus we do not know how much of the genome we recovered. However, other Dicistroviridae family was based on the complete ORF1 coding regions   Fig. . The n19dae Fig. .Figure 49 , Varroa destructor virus 1 (VDV-1) and Sacbrood virus) using Bowtie 224 and to all known viruses using Virusfinder25. To determine whether we had contamination we also mapped our negative control sequences, of which a single sequence Bowtie 2 mapped to FEX-1. Our sample sequences mapped most closely to KBV, with fewer matches to FEX-1, IAPV, ABPV, BQCV and SINV-1 . No amplification was observed in RT-PCR for ABPV, CBPV, IAPV, VDV-1, SINV-1 or FEX-1.We used qRT-PCR to screen samples for the presence of viruses we had detected using Bowtie 2 and Virusfinder, together with honey bee viruses that have been recorded previously in ants. The presence of KBV, BQCV, LHUV-1 and DWV was confirmed by qRT-PCR , was resolved as a new strain of KBV. The second matched the LHUV-1 sequence when recovered by Sanger sequencing, and the third matched BQCV. We extended the known sequence of the Argentine ant LHUV-1 virus to approximately 70% of the genome. Our qRT-PCR results detected BQCV, KBV, LHUV-1 and DWV, of which viral replication (i.e. parasitism by viruses of the ants) was confirmed for all but BQCV. A lack of observed replication for BQCV may indicate that these ants are not true hosts for this virus and it is not parasitizing them, or that the method used was not sensitive enough to detect replication at low virus titers (abundance). However, BQCV might replicate in Argentine ants under conditions or at time points missed in our survey. Viral replication does not occur at all times in all infected hosts28.The first virus was described from ants only 13 years ago4. These viruses are clearly also parasitizing Argentine ants, as replication was confirmed. While DWV has been detected in Argentine ants previously29, our study is the first record of these or any ants hosting KBV (and possibly BQCV).Our results demonstrate that populations of the invasive Argentine ant in New Zealand host a range of RNA viruses. As well as the Argentine ant LHUV-1 virus, the ants also host viruses that are associated with disease in honey bees, including KBV and DWV. These are globally widespread and important viruses. DWV, for example, has been hypothesised as the most likely candidate for the majority of global honey bee colony during the past 50 years29. In areas where these ants are associated with honey bee colonies, the honey bees appear to be infected with substantially higher DWV loads. In honey bee populations, viruses like DWV and KBV are associated with colony collapse, but only when viral load is high30. In New Zealand, another study demonstrated that the presence of Argentine ants was associated with higher DWV loads in honey bees and an average 47% mortality of hives over a 6-month period29. The same DWV strain was observed in both Argentine ants and bees. Argentine ants appear to have the dual effects of substantially elevating DWV titers in bees and raiding their hives for honey and brood29. We found that viral load of both DWV and KBV was also higher in Argentine ants when these ants were interacting with bees: markedly so for DWV, with viral concentrations five orders of magnitude higher when the ants were present in honey bee apiaries. These viruses were also present at all other sites, including in urban areas where there were no bee hives with several hundred meters of the sample collection location. Viruses such as DWV, KBV and BQCV are clearly present in these invasive ants in the absence of close contact with bees.Although the abundance of viruses  such as LHUV-1 and DWV in Argentine ant populations in New Zealand is known to vary over time, the viruses appear to be consistently present in these ant populationsDicistroviridae family. Viral infection of bacteria within Argentine ants thus seems highly unlikely. Although these viruses are clearly parasitizing Argentine ants (or possibly their endosymbionts), as shown by active replication, we do not know what their effect is on ant colonies, and studies have yet to investigate the effects of honey bee viruses on ant population dynamics. We hypothesise, however, that these viruses contribute to the population collapse of Argentine ants in New Zealand and elsewhere18. Possibly these viruses remain in a latent state until environmental or other conditions stimulate virulence. If so, it could be possible to induce virulence as a novel biocontrol, potentially by compromising the immunity of these ants.Some researches might suggest that the viruses we detected could alternatively or additionally be infecting bacterial symbionts or pathogens within Argentine ants. Bacteriophages, however, tend to belong to specific families and none have been previously found in the et al.6, Furst et al.31). DWV has previously been found in Camponotus ant species, together with IAPV6 and CBPV32, which was also hosted by the ant Formica rufa32. No ant viruses have been reported from honey bees, although this may be due to few studies specifically examining honey bees for ant viruses. To our knowledge honey bees have only been examined for LHUV-1 and SINV-333. Moreover, host specificity tests of SINV-3 have found that it does not infect ant species other than Solenopsis invicta, including closely related ant species34. Viruses continue to remain under-studied outside of model organisms35, and their prevalence within species, and degree of inter-specific transmission is still not well understood.Other studies have suggested that interactions between other host species result in honey bee viruses being shared between many Hymenopteran and non-Hymenopteran hosts 38. KBV was not detected in our earlier study on Argentine ants using RT-PCR7, although it appears from our current results to be highly prevalent, particularly where ants interact with bees. The lack of detection in earlier work may be due to the differences in the RT-PCR primers used between studies not being able to detect the proposed new strain. If this is the case, variant strains of KBV might exist undetected in ant  populations. We know that some virus strains are more virulent than others39. But we do not know which KBV strains are present in New Zealand honey bees or the distribution of the new strain globally, their relative virulence, and the degree of transmission between species.We detected a novel strain of KBV by extending a contig that previously appeared likely to be a novel ant virusAparavirus in the Dicistroviridae family, phylogenetically positioned intermediate to ABPV and SINV-1. ABPV and SINV-1 have been found to be similarly related in other work using amino acid sequences of the capsid proteins . We recovered ORF1 for LHUV-1, which included all viral non-structural coding regions. We have thus characterized the virus sufficiently to confirm its identity as a novel virus infecting Argentine ants. Our previous work has indicated LHUV-1 to be present in the ant’s home range of Argentina, and in the invaded range of Australia and New Zealand7. The virus is thus likely to be distributed throughout the ant’s invaded range.We extended the known genomic sequence of the LHUV-1 virus to cover approximately 70% of the estimated genome. Our phylogenetic analysis of both the entire ORF1 and RdRp gene clearly place LHUV-1 as an Iflaviridae (DWV6); Dicistroviridae ; and Solinviviridae . The SINV-2 virus has not been placed within a family, but like the other viruses associated with ants, it is a picorna-like virus9. Similarly, CBPV, which has also been found in ants32 is currently an unclassified ssRNA virus. Perhaps this apparent phylogenetic restriction to a few families is owing to a co-evolutionary history. However it might also be due to a lack of studies, or the ease of detection of ssRNA viruses relative to other viral groups.The ssRNA viruses that to date are known to be hosted by ants typically occur within three viral families: 23 effectively enabled us to extend the sequence length of putative novel viruses and strains. We could then use this sequence as a template for confirmation of presence via standard PCR and Sanger sequencing. Typically, RACE is used to extend these sequences . RACE can be expensive compared with standard PCR41. Thus our approach offers a cost effective alternative, particularly for narrowing the list of candidates for confirmation. While our approach enabled recovery of nearly an entire novel strain, in order to recover a full novel genome, some RACE or similar sequencing approach  will most likely be needed.We used complementary methods in our virus discovery and confirmation in order to maximise our ability to detect potential novel viruses. We found that IVA24, Virusfinder25 and MEGAN20 to identify known and novel viruses. Although Bowtie 2 and Virusfinder indicated the potential presence of more bee viruses than MEGAN, the latter results were more consistent with qRT-PCR confirmatory analysis. These differences in results can likely be attributed to the difference in analysis approaches: MEGAN analysis is based on blast results of searches on GenBank databases using assembled contigs, while Bowtie 2 and Virusfinder use individual RNA-seq reads to map entire known genomes. We therefore advise caution when using single in-silico analyses to make definitive conclusions.We used Bowtie 27 detected DWV using RNA-seq data. This virus was not observed in any assembly, or in our MEGAN analysis, or in mapping (with the exception of one single read). However, DWV has been positively confirmed in these samples via qRT-PCR here and in other species29. We know that this is not a general failure of RNA-seq to detect DWV as we detected this virus in RNA-seq data of honey bee samples that were sequenced in the same run as our ant samples, as others have also found . For the same reason this false negative result cannot be attributed to a biological feature of the virus that would make it undetectable by RNA-seq. Our qRT-PCR results showed a much lower prevalence of DWV in sites where Argentine ants were not present with bees, so it seems most likely that the virus was at undetectable prevalence in our current and earlier RNA-seq samples (which were from sites where ants and bees did not interact).One inconsistency continues to baffle us. Neither this study, nor our earlier work18. The current study and other publications demonstrate a substantial microbiota associated with Argentine ant populations43. Several of these pathogens can substantially influence the population dynamics of other social insects, such as the involvement of DWV and KBV in collapses of honey bee populations44. However, it remains to be determined if the viruses we observed have any involvement in the local collapse of Argentine ant populations in New Zealand16 or elsewhere17.Ant populations are known to undergo substantial fluctuation and even local population extinctionlater Stabilization Solution . RNA was extracted from pools of whole ants from the northern (150 ants) and southern populations (120 ants). Purification of total RNA was carried out using a PureLink RNA Mini Kit®  according to the manufacturer instructions, after homogenisation with a Qiagen Tissuerupter . RNA integrity was confirmed and quantified with an RNA 6000 Nano chip on the Agilent 2100 Bioanalyser , according to the manufacturer’s instructions. The two pooled samples together with a negative control were sequenced as 125 base paired end barcoded Trueseq libraries on an Illumina Hiseq by New Zealand Genomics Limited (NZGL) at the University of Otago. Post-processing at NZGL included quality control, excluding bases with quality scores Q > 30 and trimming of adapters. We assembled the RNA-seq data using Trinity v 2.0.619 using read normalization.We used two pooled groups of ants for the metatranscriptomic detection of viruses and discovery of potential novel viruses. In November 2014 we sampled a northern population (Auckland: three sites), and in November 2014 and June 2015 we sampled a southern population  Fig. . Ants we45. The nr and nt databases were downloaded from NCBI GenBank (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) on 1 December 2015. BLAST searches were run using an installation of BLAST v 2.2.25, on the Victoria University of Wellington Science Faculty’s High Performance Computing Facility. We used blastx searches to identify putative viral proteins. Searches of the nr database are time consuming for a large number of input sequences, and generate large amounts of data when the default xml output format is used. As we were specifically targeting viruses, and the majority of sequences were likely to be non-viral, we pre-processed the data using blastn searches of the NCBI Genbank nt databases to include sequences that matched virus and viroid taxa, which we output in tabular format. We restricted searches to a threshold e-value of 0.0001 and 90% percent identity or better to enhance search specificity. Taxonomic information was assigned to the tabular output using a custom Perl script (https://github.com/AnnaFriedlander/taxon4blast). From the resulting assignments we filtered the input data for virus taxa using a Perl script. We then used these filtered contigs as input for a blastx search of the nr database with searches restricted to a threshold e-value of 0.001, percent identity of 90, best_hit_overhang of 0.25 and best_hit_score_edge of 0.05. The blastx results were visualised in MEGAN 5.10.720. To confirm these as virus candidates we identified coding sequences of novel viral origin in our contigs by visualising our blastx results in Blast2GO21, and translating these contigs using ExPASy  to determine the appropriate reading frame.To detect potential novel viral pathogen sequences in the assemblies, we used the Trinity assembled contigs to query the National Center for Biotechnology Information (NCBI) GenBank databases using BLAST46. Reliable sequences could only be retrieved for TR30069|c0, TR30069|c1, and TR31304|c0. We translated the sequences using ExPASy and used GenBank blastp searches within MEGA7 to identify the most closely matching viral proteins.Six contigs were identified as potential novel viruses  and previously identified contigs  with the extended LHUV-1 sequence in MEGA7. We also used IVA to extend the newly identified and previously identified contigs, and Sanger sequenced those that could be extended  sequences together with sequences from GenBank and one TSA sequence predicted to be a virus of ants and other Hymenoptera . We restricted the analysis to ants and othe Hymenoptera as these groups were the closest matches to our protein sequences on GenBank. We translated the TSA sequence to obtain the RdRp region for comparison. Sequences were aligned using MUSCLE47 and phylogenetic trees were constructed in MEGA7.The alignments for both phylogenetic analyses are provided in the Microsoft Excel spreadsheet associated with Supplementary Table We translated the extended contigs using ExPASy and identified the most closely matching viral proteins with HHpred24 and Virusfinder25, which uses the nr database as a reference, to identify all known viruses. For all putative viruses detected by MEGAN, we mapped the RNA-seq reads directly to their viral genomes using Bowtie 2 to identify potential true and false positives. We then used Virusfinder to also identify potential false positives and false negatives. We then used RT-PCR to confirm the positive results from Bowtie 2 and Virusfinder. We also used RT-PCR to confirm false negatives and true negatives using selected viruses, and confirm true positives and true negatives of the viruses identified by Virusfinder and Bowtie 2 , Formica exsecta virus 1 (FEX-1),and viruses commonly infecting honey bees: DWV, Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Israeli acute paralysis virus (IAPV), Chronic bee paralysis virus (CBPV), Varroa destructor virus 1 (VDV-1) and Sacbrood virus.As well as using MEGAN to screen for all viruses, we screened our sequences for viruses known to infect bees and ants by mapping the RNA-seq reads to those virus genomes using Bowtie 2 2 Table . VirusesAdditional samples of Argentine ants were collected to both confirm the presence and compare the relative infection level of identified viruses Fig. . VirusesL. humile Ndufa8  was used to normalize the calculated viral loads48.RNA was extracted from 24–30 whole ants from each site by bead-beating  samples in GENEzol reagent  with 5% β-mercaptoethanol, with chloroform and isopropanol purification. These extractions were then pooled to provide RNA for the northern, southern locations, and where Argentine ants were co-located with bees  of 1 μg RNA was performed with a tagged forward primer at a final concentration of 100 nM (DWV Tag-F15; KBV Tag-KBV83F; VDV-1 Tag-VDVqF [as an additional negative control], or BQCV Tag-BQCV-sense; Supplementary Table 7.Viral replication was confirmed by detection of the virus negative strand using reverse transcription and RT-PCR based on the standard method for detecting DWV replicationSupplementary materials and methodsAlignments


Enterovirus 71 (EV71) is a global infectious disease that affects millions of people. The virus is the main etiological agent for hand, foot, and mouth disease with outbreaks and epidemics being reported globally. Infection can cause severe neurological, cardiac, and respiratory problems in children under the age of 5. Despite on-going efforts, little is known about the pathogenesis of EV71, how the host immune system responds to the virus and the molecular mechanisms behind these responses. Moreover, current animal models remain limited, because they do not recapitulate similar disease patterns and symptoms observed in humans. In this review the role of the host–viral interactions of EV71 are discussed together with the various models available to examine: how EV71 utilizes its proteins to cleave host factors and proteins, aiding virus replication; how EV71 uses its own viral proteins to disrupt host immune responses and aid in its immune evasion. These discoveries along with others, such as the EV71 crystal structure, have provided possible targets for treatment and drug interventions. Picornaviridae family, and notably Enterovirus 71 (EV71), coxsackie A6 (CA6) and coxsackie A16 (CA16)  is a common infection caused by a number of viruses, commonly from the 6 (CA16) . HFMD pr6 (CA16) . The mai6 (CA16) . This raThe coverage of HFMD, with the exception of Polar regions, is global in distribution . The disHand, foot, and mouth disease usually affects children between the ages of 6 months to 5 years old, as this is the time when they no longer receive the benefits of passively transferred maternal antibodies. These antibodies can aid in protecting them from the etiological agents, i.e., EV71 that cause the disease, when their own immune system is not fully developed to fight the virus on its own . After tEnterovirus A species of the Enterovirus genus of the Picornaviridae family  and Enterovirus 71 (EV71), both of which are from the e family . Other ve family . Of the  of EV71 .Enterovirus 71 has been found all over the world with different symptoms and clinical manifestations seen during different outbreaks. The virus was first isolated in California, United States in 1969 from patients suffering from central nervous system (CNS) infections with cutaneous signs . EV71 waThis protracted epidemic has drawn specific scientific interest. These outbreaks, with their larger variety of symptoms have drawn more questions that need answering about EV71 and its pathophysiology.Figure 2). P1 is cleaved into VP3, VP1, and VP0 (which is then in turn cleaved to form VP4 and VP2). The VP1, VP2, and VP3 proteins form a surface pentameric subunit together with the VP4 protein, attached to the inner surface . The capsid is formed by a quasi-T = 3 symmetry of 60 copies of this subunit to form an icosahedral capsid  and 7 non-structural proteins (2A-C and 3A-D) . The genl capsid . P2 is cB > 92.2%; C > 91.9%; D = 95.2%; E > 93.6% and F > 93.6%   . While tailable) . Furtherailable) .Figure 1; As humans are the only known hosts of EV71, all transmission is assumed to be human-to-human. The virus can survive on external surfaces for up to 3 days, so direct human-to-human contact is not always necessary as the virus can be picked up from these contaminated areas . The maiFigure 1; Figure 1; Evidence suggests that the initial phase of replication occurs in lymphoid tissues of the tonsillar crypt  and in tFigure 1; Figure 1; Figure 1; Figure 1; Figure 1). It is currently unknown what is the exact cause of cardiac failure and pulmonary oedema but is thought to be a combination of these elements, cytokine storm and brainstem destruction.It has been noted that children who had detectable viraemia after 3 days had significantly more severe complications (At least five molecules have been identified as possible cell surface receptors for EV71. Scavenger receptor B2 (SCARB2) , P-selecMouse L929 cells that normally do not support EV71 infections, became highly infected with EV71 after being transfected and overexpressed with either SCARB2 or PSGL-1 . AlthougAs there is currently no effective treatment available for EV71, understanding virus–host interactions in greater detail will provide an increased knowledge base for the successful development of medical countermeasures .The innate immune response is one of the body’s earliest ways of fighting off pathogens . This imHost cells, both immune and non-immune contain innate receptors called pattern-recognition receptors (PRRs), which can recognize materials/molecules of pathogens, and components of cell damage/death. These PRRs recognize the pathogens via the detection of pathogen associated molecular patterns (PAMPS) . PRRs caEnterovirus 71 is mainly detected via the RLRs; RIG-I, melanoma differentiation-associated protein 5 (MDA5), and TLR3 . The binin vivo and patient studies suggest that IFN’s offer little help as a treatment after contraction of the virus (Interferons (IFN’s) are molecules that are vital to the body’s immune response in order to help control virus replication and spread . Based ohe virus .Figure 2; Table 1). This cleavage diminishes the production and activation of IRF3 and IFN type I  . This teTable 1; Some novel ITAFs for EV71 have also been discovered. Far upstream element binding protein 1 and 2 (FBP1 and 2) have both been identified as novel ITAFs for EV71 . FBP1 enEnterovirus 71’s ability to interrupt, intercept and disrupt the host immune response is critical to the survival and propagation of the virus, and whilst this has a negative effect for the patients, it gives an insight into possible areas that could be targeted to disrupt virus biology.In vitro models whilst being the initial “go to” model for viral experiments, can come in many different forms and serve many different purposes. The most basic of these are isolation and viral kinetics, for which rhabdomyosarcoma (RD) cells are the most utilized for EV71  or Jurkat (T cells) these issues can be easily over come. THP-1 cells have been used to show the role of NLRP3 inflammasome activation and IL-1β maturation during interaction with the EV71 protein 3D   .in vivo mouse models of EV71 have demonstrated an age-dependent susceptibility to the virus  EV71 experimental infection using older mice. Therefore, this hybrid model lends a strong support to the importance of both entry factor and host innate immunity .Many drugs that have been already developed and used to fight other picornaviruses, such as polioviruses and rhinoviruses, were tested for their abilities to limit EV71 infection. These drugs, ribavirin , pleconain vitro inhibition of EV7  showed potent neutralizing capabilities in vitro . It was in vitro .Antibodies raised in mice against a conserved VP3 knob region have shown protective neutralizing effect across the different subgroups of EV71 . Whilst After the success of the polio vaccines, it was envisioned that EV71, closely related to polio, would also be an easy target for near eradication via a vaccine. Studies in mice demonstrated that the transfer of antiserum provided protection against EV71, further indicating the attainability of a vaccine . EV71 vaLive attenuated vaccines have been tested in cynomolgus monkeys and these induced the production of high levels of neutralizing antibodies that provided cross protection across the sub groups . HoweverDNA vaccines have been attempted for EV71. However, DNA constructs containing EV71 VP1 gene that elicited specific VP1 immunoglobulin G’s (IgG’s) and neutralizing antibodies conferred low levels of antigenicity .Inactivated whole virus vaccines have been shown in mouse models of EV71 to produce very high levels of virus specific antibodies that are cross neutralizing . These sThere have been major breakthroughs in EV71 research in the last couple of decades: from the identification of host receptors, to the discovery of molecular mechanisms of virus–host interactions, to the phase III trials for vaccines. Although there is an understanding of EV71 pathogenesis, many areas of uncertainty remain with many mechanisms still not well understood and many pathways that have yet to be proven functionally. There is still a need for an animal model that better mimics the human disease. The development of such models will enable us to gain a much better understanding of the path of EV71 infection, and may give us an insight in to new molecular mechanism that we can target to interrupt the virus and stop the infection.JC searched the literature and compiled and wrote the review. LN helped design, write, and edit the review. JH, TS, and M-HO helped to write, edit, and critique the review.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.


Consumption of sugar causes tooth decay, overweight and obesity related morbidities. This paper in response to the Minister of Health’s request, provides estimates of the mortality, morbidity and health care costs attributable to sugar consumption in Israel along with the effects of reducing sugar consumption.Gender specific relative risks of many diseases from overweight (25 < =BMI < 30) and obesity (BMI > =30) were applied to the national gender specific prevalence rates of overweight and obesity in order to calculate the population attributable fraction (PAF) from overweight and obesity. National expenditure on these related diseases was calculated by applying disease-specific data from a recent Canadian study to estimates of disease specific general hospital expenditures in Israel. Disease specific costs attributable to overweight and obesity were estimated from the product of these expenditures and PAF. In addition national costs of treating caries in persons under 18 years of age from sugar were calculated. Similar calculations were made to estimate the burden from sugar in terms of mortality and hospital utilisation. A recent UK modelling study was used to estimate the effect of a national program to reduce calorific consumption of sugar from 12.45 to 10% in 5 years.Conditions associated with overweight or obesity accounted annually for 6402 deaths (95% CI 3296–8760) and 268,009 hospital days. Dental costs attributable to sugar consumption were 264 million NIS. In total, obesity, overweight and sugar consumption accounted for 2449 million in direct treatment costs (0.21% of GDP), rising to 4027 million (0.35% of GDP) when indirect costs were included. A national program of reducing energy from sugar consumption from 12.45 to 10% over 5 years is considered have a very feasible short-term goal. Even if the program does not impose taxes on sugar consumption, this would save 778 million NIS as well as 1184 lives.Sugar consumption causes a huge monetary and mortality burden. Estimates of potential decreases in this burden justify the current prioritisation given by the health minister of creating and implementing a national program to reduce sugar consumption, which is likely to be cost-saving (ie: averted treatment costs will exceed intervention costs). The increasing prevalence of obesity, which has more than doubled since 1980 . In 2000Overweight and obesity, as well as their related non-communicable diseases, are largely preventable. It is acknowledged that there exists a multiplicity of measures to prevent overweight or obesity such as encouraging physical activity , reducinLimit their energy intake from total fats and sugars ;Increase their consumption of fruit, vegetables, legumes, whole grains and nuts Engage in regular physical activity , 3.At the individual level, people canIndividual responsibility can only have its full effect where people have access to a healthy lifestyle. Therefore, at the societal level it is important to undertake policies that support individuals to engage in regular physical activity and make healthier dietary choices .A gradual reduction of sugar content in everyday food and drink products, combined with reductions in portion size.Price increases of a minimum of 10–20% on high sugar products through the use of a tax or levy such as on full sugar soft drinks .Reduction in price promotions in all retail outlets including supermarkets, stores, restaurants and takeaways.Reduction in advertisements for high sugar food and drink products to children and adults.Implementation of public sector catering standards to ensure provision and sale of healthier food and drinks in hospitals, leisure centers etc.Encourage health promotion by providing practical steps to help individuals lower their own and their families’ sugar intake.Consuming too much sugar and too many foods and drinks high in sugar content, causes not only tooth decay  but alsoJust as in other developed and developing countries, Israelis are not immune from being overweight or obese and their related morbidities .This paper is a response to the request of the Minister of Health, to provide an estimate as to the potential reduction in mortality, morbidity and health care costs if a national program (containing many elements similar to that proposed in the UK ), but wiEstimates of the gender, religion (Jews and non-Jews) and age-specific  measured prevalence rates of overweight and obesity in Israel in 2016, were based on extrapolations and interpolations of data from a number of national surveys on self-reported –14 and mCause and gender-specific data on relative risks  for 16 diagnoses were obtained from a Canadian cost of illness study based on meta-analyses  and fromThe gender and diagnosis specific PAF, were calculated, for both the prevalence of overweight and obesity in turn using the following standard formula:-The resultant PAF were then aggregated across genders and combined for overweight and obesity in order to arrive at a composite diagnosis specific PAF.Age, gender and cause specific data on days hospitalization in general hospitals for persons over 20 in 2016 were estimated by applying the average age, gender and cause specific hospitalization rates in persons aged 20+ from 2010 to 2013  to the age and gender specific population data for 2016, which in turn was based on extrapolations of data from 2013 to 2015 –24. TotaCause specific costs of out of hospital pharmaceutical use and other services  were estimated by applying cost ratios from a Canadian study  to the IEstimates of indirect costs included mainly productivity losses, due to increased absenteeism and presenteeism , in addition to informal carers costs and transport costs. Imputed human capital costs from premature mortality were excluded as these are in effect “virtual” costs, but frictional employment costs  were included. Estimates of the ratio of indirect costs relative to health service costs were based on international published literature , 27–41, The disease-specific product of these expenditures and PAF estimated the various cost of these diseases.Israelis consume on average 22.9 kg per capita (62.7 g. per day) of sugar annually , this prThe following formula was used for milk and adult teeth in turn, aggregated up to 18 years old:-Assuming fluoride in the water supply reduces caries by 35.5% (95% CI 25.7–45.3%) in milk teeth and 26.4% (95% CI 16.4–36.6%) in adult teeth .Cost of fillings were based on sick fund specific costs (ranging from 162 to 193 NIS for a single surface and 253–596 NIS for multiple surfaces) in proportion to their membership  and privGender and cause specific data on mortality for persons over twenty years old in 2016 were estimated by applying the average age, gender and cause specific mortality rates in persons aged 20+ from 2009 to 2013  to the aforementioned age and gender specific population data for 2016 –24.These cause and gender specific deaths were then multiplied by the cause and gender specific PAFs in order to estimate the number of deaths attributable to overweight and obesity in 2016.Extrapolations of age and gender specific life expectancies to 2016 , 23 wereAge- and gender-specific disability weights, used by the Ministry of Health, were applied to the life expectancies in 2016 , 23 in oDue to lack of cause specific disability weights, we assumed that each days hospitalization resulted in a disability weight of 0.3 , which was then multiplied by the age-and gender specific hospitalization rates adjusted by the Ministry of Health’s age- and gender-specific disability weights of health people in order to estimate the DALY losses due to morbidity.Data from a recent UK modelling study  was usedThe basic formulae used for calculating the cost per averted DALY loss is:-where:-From the health services perspective:From the Societal perspective:All costs are presented in new Israeli shekels (NIS) at 2016 price levels. (at an exchange rate of 3.86 NIS to US dollar). Israel has no official policy guidelines for defining whether or not a health intervention is cost-effective. Affordability of interventions is a function of the amount of resources available in a country as proxied by its Gross Domestic Product (GDP) per capita. Therefore, WHO guidelines were used, which define an intervention as being very cost-effective  if the cEstimated sugar tax revenues were based on recent UK estimates of 520,000,000 lb sterling per annum from just taxing soft drinks  adjustedTable Disease specific relative risks for overweight and obesity are especially elevated for type II diabetes (both genders), pulmonary embolisms (both genders), osteoarthritis  and coronary arterial disease  Table .Table 2DEstimated direct costs of comorbidities related to overweight and obesity in Israelis aged over 20 years old were around 4763 million NIS of which hospital care accounted for 44.8% and pharmaceutical consumption outside of hospitalization 24.2% .Since the PAF for Type II Diabetes exceeded 100%, we constrained its PAF to be an arbitrary 90%. Around 6402 people 95% CI 3296–8760) died in 2016  or 7.1 (discounted) years of HALE, with an average associated disability weight (DW) of 0.43, which reflects the high percentage of deaths in the 75–84 (25%) and 85+ age groups (41%) when functional deterioration has set in even amongst healthy persons.Around 268,206  hospital days were attributable to Overweight  and Obesity , accounting for 16.1% (95% CI: 4.5–23.7%) and 27.6% (95% CI: 17.5–34.0%) respectively of the 612,844 hospital days associated with related diagnoses .Coronary Artery Disease (21.5%) Type II diabetes (21.1%), and Hypertension 20.4%) account for over three-fifths of the 2185 (95% CI: 878–2667) million NIS direct costs , giving an estimate of 1578 (95% CI: 977–2841) million NIS for the indirect costs. Therefore, the total costs to society attributable to overweight (1381: 95% CI 74–2581 million NIS) and obesity (2382: 95% CI 1197–3554 million NIS), amount to around 3763 (95% CI: 3163–5027) million NIS annually.The estimate of caries prevalence per person attributable to sugar consumption was 1.25 (in children aged 6.5 years) and 2.51 (in children aged 12.5 years). This translates into an annual per capita caries incidence attributable to sugar of 0.21 for milk teeth and 0.39 for adult teeth respectively . There iHence, the direct and societal costs attributable to obesity, overweight and sugar consumption are 2449 million and 4027 million NIS respectively (both costs including 264 million caries costs attributable to sugar consumption), representing 0.21 and 0.35% of GDP respectively.In contrast to deaths which are clearly attributable to a given causality , deaths due to pollution and to personal behaviours, such as smoking, sedentariness and nutritional habits are harder to identify. Despite this difficulty, obesity and overweight have been implicated as risk factors for many causes of death , 49, 50.The estimate of 6402 deaths annually attributable to overweight and obesity  is over 17 times the magnitude of suicides, 18 times that of road accident fatalities and around 50 times that of homicides in Israel . The dirThe total costs to society of treating morbidity from obesity and overweight, is around 3.8 billion NIS, equivalent to 0.32% (95% CI: 0.27–0.43%) of GDP. , 18, an An as yet undefined, national program of reducing energy from sugar consumption from its current level of 12.45 to 10% will reduce damage from sugar related obesity (and overweight) by 3.7% over a five year time horizon [If a more ambitious target of reducing consumption to 5% is achieved over a 15 year period, sugar related obesity (and overweight) will decrease by 7.7% [Assuming the imposition of an intervention program will succeed in reducing energy consumption from sugar to 10% over 5 years, there will be a saving of 1184 lives and 14,703 PYLL, giving a total of 8425 averted discounted DALYS. Since 99.7% of these DALYS are due to mortality gains, varying the disability weight of hospitalization between 0.2 and 0.4 will have an insignificant effect on the results.Imposing a tax on sugar products could be part of the multifaceted approach to reducing sugar consumption. Taxing soft drinks could annually generate around 280 million NIS of income (based on adjusted UK estimates). In the UK such revenues are earmarked to improve primary school sports facilities. In Israel, around 24 million NIS of the revenues annually could be earmarked to add 100 full time equivalent dietician posts (including office facilities) to help implement other parts of the multifaceted program to reduce sugar consumption. Over 5 years this hypothetical intervention will for an intervention cost of around 120 million NIS, generate 1400 million in tax revenues, save 606 million NIS in direct treatment costs and a further 292 million in indirect costs, resulting in a net (cost-) saving of 2178 million NIS.However, the Health Minister is reluctant to impose a sugar tax, on the grounds that such a tax will raise consumer prices in a regressive fashion . In thisi)Israeli-based cause and age-specific relative risks, thus allowing calculation of PAF due to obesity and overweight in Israel.ii)Israeli based disease specific costs and utilization rates of geriatric hospitals, out of hospital pharmaceutical and other care costs .iii)Israeli-based cause specific estimates of work productivity losses and other indirect costs.iv)Estimates of disability weights relating to morbidity in an out-of hospital setting. This would correct for the underestimation of DALYS averted (and subsequent overestimation of Costs per DALY averted) since only estimates for morbidity losses during the period of hospitalisation were included in the model.These estimates, of damage from sugar consumption, should be regarded as preliminary as they can be improved upon if the following data ever becomes available:Gains from reduced caries might also be overestimated to the extent that some people in Israel are already exposed to fluoride via toothpaste and their natural water supplies. On the other hand, estimates of the costs of treating caries and hence potential savings attributable to reductions in sugar consumption can be considered as being very conservative as they do not include any costs incurred by persons aged 18 years and over, who will almost certainly benefit from a reduction in sugar consumption .Further underestimations of averted DALYS and averted treatment costs arose because the model was based solely on the relative risks of overweight and obese persons and was unable (because of lack of data) to take into account the impact of reductions in sugar consumption in persons of normal weight. For example, many new onset cases of type II diabetes occur in persons of normal anthropometric proportions who would also benefit from reduction in sugar consumption .All the estimates are subject to the important caveat that any achieved calorific reduction due to decreased sugar consumption is not compensated for by increased calorific intake of other (non-sugar) foods. But even in the event that a compensating rise in calorific consumption occurs , some health benefits are likely to still be generated as “not all calories are equal” since there is evidence that the quality of fat and carbohydrate can play a more important role than the quantity . The resReducing energy from sugar consumption from its current level of 12.45 to 10%, over a five year time horizon is considered to be a very reasonable and attainable short-term goal. Over these 5 years, this would save 2178 million NIS in costs (778 million NIS if no taxes are imposed) as well as 1184 lives. Achieving a reduction to 5% over a 15 year period would be a far harder goal to achieve, but the rewards in terms of decreased mortality, morbidity and expenditures would be greater.It is highly likely that any package of interventions, with or without the imposition of taxes on sugar, will be cost-saving , since costs savings from morbidity reductions (and possible tax revenues) will exceed the costs of the intervention. This will still be true if we view the results just from just the direct costs of health services perspective. Finally, it should be noted that other many other interventions available to reduce overweight and obesity are available, that have also been shown to be cost-saving or very cost effective , 56.Sugar consumption causes a huge monetary and mortality burden. Estimates of potential decreases in this burden justify the current prioritisation given by the health minister of creating and implementing a national program to reduce sugar consumption, which is likely to be cost-saving (ie: averted treatment costs will exceed intervention costs).


PPR) is an economically important disease of small ruminants with a rapidly expanding geographical distribution. Peste des petits ruminants virus may manifest in a variety of ways with disease ranging from acute to subclinical. We investigated the exposure of large ruminants to PPRV in areas where the virus is endemic in the small ruminant population by assessing the serological status of groups of animals. This study focused on the Punjab province of Pakistan as an area where the virus is endemic and where mixed farming practices occur enabling close interactions between small and large ruminant populations. An overall PPR seropositivity was detected in 10.0% of cattle and 14.16% of buffaloes. Following an assessment of serological profiles in large ruminants within different age groups, a maximum seroprevalence was observed in cattle (17.5%) and buffaloes (22.5%) over 2 years of age indicating the potential utility of sampling large ruminant populations for PPR serosurveillance. The large ruminants sampled between one and two years of age had similar levels of seropositivity within populations with 11.2% and 16.2% of animals being seropositive, respectively. Current PPR vaccination strategies do not enable the differentiation between infected and vaccinated small ruminants, and as such, the serological surveillance of sheep and goats is of little value. When considering eradication programmes for PPRV, this factor is of great significance. However, where large and small ruminants are farmed together, serological surveillance of large ruminants may provide a snapshot of virus infection within populations where mild disease is present or where small ruminants are regularly vaccinated.Peste des petits ruminants ( Morbillivirus in the family Paramyxoviridae alongside measles virus (MV), rinderpest virus (RPV), canine distemper virus (CDV), phocine distemper virus (PDV), porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and recently identified novel morbilliviruses infecting cats (FeMV), bats and rodents  is a highly contagious and acute viral disease of sheep and goats that causes great economic losses due to the high levels of morbidity and mortality often observed. The causative agent, PPR virus (PPRV), belongs to the genus Following rinderpest eradication, measles and PPRV have also been identified as targets for potential global eradication. Indeed, capitalizing on the successful eradication of rinderpest, various international agencies including United Nations Food and Agriculture Organization (FAO) and the World Organization of Animal Health (OIE) are now focusing on PPR as the next veterinary virus for potential eradication. For any disease eradication programme, epidemiological surveillance is critical .Blood samples were collected from animals from 10 districts within the Punjab province of Pakistan between June and December 2009 Table , 2009. HFollowing sampling, the serum samples were divided according to the age of the animals into the following groups: (i) young animals less than one year of age, (ii) animals between one and two years of age and (iii) animals over two years of age.Each sample was tested for the presence of PPRV‐specific antibodies using the anti‐hemagglutinin (H) PPRV competitive ELISA . The assays were performed and analysed following the manufacturer's instructions. Samples with percentage inhibition value >50% were considered positive as per Anderson and McKay . All theThe main aim of this study was to investigate the seroprevalence of PPRV in cattle and buffalo populations in an agro‐ecologically defined area of Punjab in Pakistan where PPR is endemic in small ruminant populations. The presence of PPRV‐specific antibody was demonstrated in both cattle and buffalo and is proposed as an indicator of virus circulation in areas where large and small ruminants are farmed together. This highlights a potential mechanism to screen large ruminant populations to determine the asymptomatic circulation of PPRV in small ruminant populations both in the presence and absence of local vaccination. This mechanism of screening large ruminants may aid efforts to detect circulating PPRV in the absence of a DIVA vaccine for PPRV, especially where coordinated vaccination campaigns have been initiated. Certainly, if regular vaccination is implemented in sheep and goats, serosurveillance of domestic large ruminants gives added value to routine PPR clinical surveillance not only in Pakistan, but also in many other PPR endemic countries where mixed farming practises are implemented. Alongside serological assessment, monitoring of large ruminant species in areas where PPRV is endemic in small ruminants may indicate any potential disease profiles that may manifest should PPRV evolve to cause clinical disease in these species. Transmission of PPRV from infected goats to cattle has been recently reported . Peste des petits ruminants outbreaks are generally seen to cause mild to moderate disease in sheep and goats in Pakistan due to the endemic situation. There have been occasional outbreaks of severe disease within Pakistan and although the drivers for differences in pathogenicity following infection with PPRV are ill‐defined, the introduction of virus to populations of naive animals often results in severe clinical disease.The detection of serological evidence for PPRV infection of large ruminants across the districts was evaluated Table . A largeAs shown in Table In this study, to validate the serological positivity detected in large ruminant samples, 200 bovine sera from Italy were tested on the cH‐ELISA. All 200 samples were negative demonstrating a lack of false positivity seen with the assay when testing large ruminant sera. Whilst no test can be truly 100% specific, if the sample size is large enough, false positives may be detected. Indeed, the specificity of this test was at least as high as that described for the RPV‐based assay (99.9%). A similar level of specificity was reported by Couacy‐Hymann et al. , using tWhilst the serological positivity of large ruminants for PPRV has been reported previously where outbreaks in small ruminants have occurred  and buffalo  were observed.The findings in the present study largely agree with previous reports regarding the seropositivity of large ruminant populations to PPRV. Certainly, the observation that in general, buffalo seroconvert more readily than cattle is reflected in several published manuscripts (Khan et al., In conclusion, the assessment of serological profiles of large ruminants in areas where small ruminants have been vaccinated against PPR may serve as a useful tool for the detection of circulating PPRV in the absence of DIVA vaccines. Across many areas considered endemic for PPR, large and small ruminants are housed and reared together creating numerous opportunities for virus transmission.


The emerging disease of rabies was confirmed in Taiwan ferret badgers (FBs) and reported to the World Organization for Animal Health (OIE) on July 17, 2013. The spread of wildlife rabies can be related to neighborhood countries in Asia. The phylogenetic analysis was conducted by maximum likelihood (ML) methods and the Bayesian coalescent approach based on the glycoprotein (G) and nucleoprotein (N) genes. The phylogeographic and spatial temporal dynamics of viral transmission were determined by using SPREAD, QGIS. Therefore, the origin and the change with time of the viruses can be identified. Results showed the rabies virus of FB strains in Taiwan is a unique clade among other strains in Asia. According to the phylogeographic coalescent tree, three major genotypes of the FB rabies virus have circulated in three different geographical areas in Taiwan. Two genotypes have distributed into central and southern Taiwan between two ecological river barriers. The third genotype has been limited in southeastern Taiwan by the natural mountain barrier. The diversity of FB rabies viruses indicates that the biological profile of FBs could vary in different geographical areas in Taiwan. An enhanced surveillance system needs to be established near the currently identified natural barriers for early warnings of the rabies virus outbreak in Taiwan. However, in June 2013, after ruling out the possibility of canine distemper and pseudorabies infections in three dead ferret badgers  from Nantou and Yunlin Counties and using a series of tests for rabies diagnosis at the National Taiwan University, including Immunohistochemistry (IHC) and RT-PCR, the first rabies case was finally confirmed on June 26, 2013 by the National Laboratory, Animal Health Research Institute, Council of Agriculture, and Executive Yuan, Taiwan. The results were reported to the World Organization for Animal Health (OIE) on July 17, 2013, and Taiwan was declared as a rabies-infected area.Taiwan had been recognized as a rabies-free area since 1961 . Although several wildlife species have been shown to be the main reservoirs of rabies around the world, the role of FBs in rabies transmission to dogs and humans has been identified only in Mainland China [A variety of island-wide wild species especially carnivores have been reported exhibiting aggressive behaviors, neurologically ill, and dead. Among more than hundreds of carnivores tested so far for rabies, confirmed rabies cases were almost exclusively (99%) identified in ferret badgers (FBs). In a surveillance report for 1016 wild carnivores, 33.2% from 831 Formosan FBs were fluorescent antibody test (FAT) positive. Most of other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative [Although a surveillance system for canine rabies has been established in Taiwan for many years, the unique rabies viral strains found in FBs in this study seem to have been silently circulating in this animal species but without spillover to domestic dogs or cats in Taiwan for a long period of time. How this rabies virus strain was introduced in Taiwan and the main reason for the emergence of this outbreak are still unknown and need further investigation. This incidental discovery of wildlife rabies in Taiwan raised two major questions: was the emergence of wildlife rabies due to the accidental introduction of the virus through illegal animal importation from neighborhood countries in Asia? Or was the disease endemic in FBs but went undetected since dog rabies was eradicated in Taiwan? This study was thus conducted to address these issues.http://www.nvri.gov.tw/Module/NewsContent/400/392.aspx?nid=%2bp7kpxBWWe0%3d&type=zO4l76wykT8%3d; 2013/11/15; GenBank Accession number: KF501174.1-KF501185.1). These sequences were compared to sequences of 75 glycoprotein and 63 nucleoprotein of rabies viral strains in other countries, including China, Philippines, Thailand, Vietnam, India, Africa Korea, Laos, Cambodia, Nepal, and USA .Phylogenetic analysis by maximum likelihood (ML) methods and coalescent analysis Figures  and 2 wehttp://www.megasoftware.net/) was used for ML analysis. The maximum clade credibility (MCC) tree of these two proteins was constructed by BEAST version 1.7.5 (http://beast.bio.ed.ac.uk), which implements a Bayesian coalescent approach through Markov-Chain Monte Carlo method for 80 million samplings. The estimated time to most recent common ancestor (TMRCA) and its 95% highest posterior density (HPD) values were obtained.The software of MEGA version 5.2 (http://www.phylogeography.org/SPREAD.html) and google earth ArcGIS was constructed for understanding if the viral evolution could be associated with ecological barriers. Posterior distributions under the Bayesian phylogeographic model [The phylogeographic tree by SPREAD 1.0.6 , 6 civet cat (1.1%), 1 shrew, and 1 dog. The geographic distribution of those animals with Taiwan rabies viruses had sporadically shown the high mountain areas still had few infected shrews, FBs, and also civet cats . Most ofOur results further indicated that the substitution rates of FB rabies viruses were similar in central and southern Taiwan. The observations may imply a similar ancestral history of rabies virus lineages in central and southern Taiwan. After the rabies virus adapted to a new host species, rates of viral evolution may be more likely affected by biology of the maintenance host rather than rabies virus itself . Therefo (Mustela putorius furo) showed a low isolation rate of rabies virus from the salivary glands of the ferrets after inoculation of rabies viral strains from naturally infected ferrets [The long term existence and unique strains of FB rabies viruses found in different geographical areas in Taiwan, which is similar with previous studies , 6, 7, m ferrets . For eff ferrets . The hig ferrets  also impThe results shown by the present phylogeographic analysis support the idea that the three unique rabies viral genotypes circulating in FBs could be related to geographical barriers in Taiwan. The similar findings have been shown in previous studies to understand evolution of dog rabies viruses , 10. AftTaiwan rabies from FBs also presented the division by geographical region , 7. The Moreover, ecological studies on population density, seasonal movement, and eating behaviors of FBs in Taiwan are necessary to understand the implications of these factors for rabies transmission among these animals, the interaction of FBs, and other kinds of wildlife living in the same environment. This information will be helpful to establish a more efficient surveillance system for understanding the dynamic changes of rabies epidemics in Taiwan. Also, the results can be applied for the development of effective oral rabies vaccines for rabies control among wild ferret badgers in Taiwan. Moreover, the infectious case of rabies virus in human has not been found in Taiwan area. Therefore, further virulence testing is suggested for the degree of cross-species transmission.The background information of glycoprotein (G) and nucleoprotein (N) reference sequences of the rabies viral strains using in this study for phylogenetic data analysis presented the GenBnak accession number, country source and the year of isolation. The 75 glycoprotein and 63 nucleoprotein of rabies viral strains came from China, Philippines, Thailand, Vietnam, India, Africa Korea, Laos, Cambodia, Nepal, and USA.


Caedibacter taeniospiralis is an obligate endosymbiont living in the cytoplasm of Paramecium tetraurelia. C. taeniospiralis causes the so-called killer trait, eliminating intraspecific competitors of its host when released into the medium by the concerted action of the unusual protein structure R-body (refractile body) in addition to an as-yet-unknown toxin. Caedibacter taeniospiralis  lives in the cytoplasm of its host, Paramecium tetraurelia (Ciliophora). This unicellular, relatively large (150-µm) eukaryote separates vegetative and sexual functions, possesses germline and somatic nuclei, and is a long-established model in genetics and epigenetics research   . This stle body) . Caedibain situ hybridization, which enabled detection of intracellular C. taeniospiralis 51T, as described elsewhere (P. tetraurelia cultures (strain 51K = CCAP 1660/3F) were fed with β-lactam-hypersensitive Escherichia coli ΔtolC , including the previously known plasmid, pKAP, of 41.7 kb (Total DNA was isolated from exponentially growing paramecia. Their infection status was verified by fluorescence lsewhere . To minisolation . We genesolation , 24 contt of 28% . Draft g 41.7 kb . The genGene annotation of the draft sequence was carried out using Prodigal , tRNAscaC. taeniospiralis strain 51K will improve our understanding of this organism and its symbiotic interaction with P. tetraurelia, including and in addition to the killer trait. The availability of this sequence information enables phylogenomic analyses of the genus Caedibacter and will provide a valuable resource for the identification and analysis of toxin candidates, which might have unusual biological properties that explain the need for the R-body as a delivery device.The genome sequence of PGGB00000000. The version described in this paper is version PGGB01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients  were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections. Case-patients were from the United States , Brazil (n = 7), and Colombia (n = 7). Tested specimens were placenta, umbilical cord, fetal tissues (from pregnancy loss) from 44 women suspected of being infected with Zika virus during pregnancy, and different portions of the brain , kidney, liver, spleen, lung, and heart from 8 infants with microcephaly who died. We also included in this analysis all available clinical, demographic, and travel history information and other relevant laboratory results from the state and local health departments and the CDC National Center for Emerging and Zoonotic Infectious Diseases, Division of Vector-borne Diseases, Arboviral Diseases Branch (including available serology and serum RT-PCR results). Clinical status of the infant, including determination of microcephaly or apparently healthy status, was based on the information  provided to CDC by the state and local health departments and referring clinicians or pathologists as of date of testing. All samples and associated medical and autopsy records were provided in the context of diagnostic consultation, a routine public health service provided by CDC. As such, institutional review was not required for the testing described in this article. Clinical and pathologic findings for 5 cases from Brazil have been previously described  for whom Zika virus infection was clinically and epidemiologically suspected. The tissue specimens were submitted during December 2015–July 2016 to the Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Division of High-Consequence Pathogens and Pathology, Infectious Diseases Pathology Branch , by local and state health departments and pathologists for diagnostic consultation. In this series, the definition of a case-patient was 1) a pregnant woman with possible Zika virus disease . To evaluate the level of fragmentation and presence of PCR inhibitors, we also tested each sample by housekeeping gene 18S rRNA RT-PCR by using QuantumRNA Classic 18S Internal Standard .We designed 2 sets of primers that target the nonstructural 5 (NS5) and envelope (E) genes of Zika virus and developed RT-PCR assays for the detection of Zika virus RNA from FFPE tissues. We validated the RT-PCR assays by using various positive and negative controls. Positive controls were RNA extracted from FFPE blocks of cultured cells infected with Zika virus prototype  and Brazil 2015 strains. Negative controls were RNA extracted from FFPE cell culture controls or tissue specimens from persons with previously confirmed infection with the following viruses: dengue types 1–4, West Nile, yellow fever, Japanese encephalitis, St. Louis encephalitis, eastern and western equine encephalitis, chikungunya, herpes, parvovirus B19, cytomegalovirus, adenovirus, enterovirus, rubella, Powassan, and Lacrosse. We extracted RNA from FFPE tissues of all 52 case-patients (multiple FFPE tissue blocks per patient) by using an optimized extraction protocol as previously described  were also used as negative control probes for ISH. To localize Zika virus genomic RNA (using antisense probe) and negative-sense replicative RNA intermediates (using sense probe) in tissues, we performed ISH on FFPE brain and placental tissues positive for Zika virus by RT-PCR, as previously described , glial fibrillary acidic protein , and CD163  on serial sections of block positive for Zika virus by ISH from selected case-patients, according to previously described protocol . ISH was developed and validated on Zika virus–positive culture cells and on various Zika virus–negative controls, including tissues from case-patients or cultures positive for dengue virus, West Nile virus, and chikungunya virus. Riboprobes targeting dengue virus and Statistical analyses were performed by using GraphPad Prism statistical software, version 6.0a . We compared demographic and clinical variables between the 2 groups by using the Fisher exact test (2-sided). We used the Mann–Whitney U test for 2-group comparisons of continuous data. Differences were considered statistically significant at p<0.05.We identified Zika virus RNA by RT-PCR in various tissue specimens from 32 (62%) case-patients . Median Brain and placental tissues from 32 (62%) of the 52 case-patients were positive by both Zika virus RT-PCR assays (gene targets NS5 and E). Sequence analysis of all positive amplicons showed 99%–100% nt identities with Zika virus Asian genotype strains currently (2015–2016) circulating in Brazil. RT-PCR results were positive for 24 (75%) of 32 case-patients with adverse pregnancy (n = 12) or birth (n = 12) outcomes and positive for 8 (36%) of 22 case-patients with live-born, apparently healthy, infants (p = 0.0082). Of 24 case-patients with positive RT-PCR results and adverse pregnancy/birth outcomes, 23 had maternal symptom onset during the first trimester, whereas all 8 case-patients with apparently healthy infants and positive RT-PCR results had symptom onset in the third trimester (p<0.0001).Of the 13 microcephaly-associated case-patients , 8 were The time frame from maternal symptom onset to detection of Zika virus RNA by RT-PCR in brains was 119–238 (mean 163) days and in placentas was 15–210 (mean 81) days. Relative levels of Zika virus RNA in the infant brain tissues  were ≈1,Overall, Zika virus RNA was demonstrated by ISH in tissues of 16 (50%) of 32 case-patients with positive Zika virus RT-PCR results. Intense signals from antisense (which binds to Zika virus genomic RNA) and sense (which binds to replicative RNA intermediates) probes were observed in brain tissues  of 7 of This work demonstrates evidence of Zika virus RNA replication in placentas of women who had pregnancy losses during the first or second trimester of pregnancy and in brain tissues of infants with microcephaly. We directly localized Zika virus negative-sense replicative intermediates in Hofbauer cells of placenta and neural cells and neurons by using ISH. Our findings indicate that Hofbauer cells may play a role in the dissemination or transfer of Zika virus to the fetal brain, particularly during early pregnancy. Furthermore, tissue-based RT-PCR assays described herein also extend the time frame for Zika virus detection; thus, the assays can be a valuable adjunct for the diagnosis of congenital and pregnancy-associated infections and can help to expand diagnostic opportunities for Zika virus, particularly when the mother was not previously tested, the window of detection for serum RT-PCR and serology has passed, or results of Zika virus testing are inconclusive . In addition, our findings also reveal the persistence of Zika virus RNA in placenta and brain tissues, which might provide insights into the potential late or long-term sequelae of the infection.–,. Furthermore, we noted that the relative levels of Zika virus RNA in the brain tissues of infants were >1,000-fold higher than those in placentas, according to real-time quantitative RT-PCR, which also suggests replication of Zika virus in brain. Previous case reports describe identification of Zika virus antigens and RNA predominately in fetal brain tissues by IHC and RT-PCR  has been reported  of 14 women who had spontaneous abortions or fetal losses. All of these women had symptom onset during the first trimester. For 9 of these 12 case-patients , replicative Zika virus RNA was demonstrated in Hofbauer cells  of chorionic villi, suggesting direct infection and replication of Zika virus in Hofbauer cells. We have previously reported the presence of trophoblast necrosis and fibrin deposit along with viral antigens in placenta, which may also indicate placental damage by direct infection  was also detected in placental tissues of 8 women who had apparently healthy infants. In each of these women, symptom onset began during the third trimester; for 5 of them, serologic evidence of Zika virus or unspecified flavivirus infection was found. However, in 3 apparently healthy infants, serum/cord blood RT-PCR or serology results were negative for Zika virus or flavivirus; for remaining 5 infants, Zika virus testing was not performed. Detection of Zika virus RNA in the placenta by RT-PCR cannot distinguish between maternal and fetal infection. The absence of apparent abnormalities in these infants could be because 1) Zika virus may not have transferred from the mother to the fetus in utero because the late-pregnancy placenta might have protected the fetus, or an effective fetal immune response might be present; or 2) the critical period of organogenesis was complete before maternal or possibly fetal infection; therefore, no apparent/major malformations were identified at the time of birth. Clinical implications for an infant with Zika virus RNA detected in the placenta, in the absence of laboratory evidence of Zika virus in the infant, are unknown. However, the negative Zika virus testing results (cord blood or serum RT-PCR and serology) in these infants should be interpreted with caution; the results could be negative because the window of Zika virus detection might have passed. Periodic monitoring of these infants may be helpful for early recognition of potential late sequelae of congenital infections. Two recent studies observed neurologic abnormalities in infants whose mothers acquired Zika virus infection in the third trimester of pregnancy; for 1 of these infants, neurologic abnormalities were first identified at 6 months of age (In conclusion, our findings further support the linkage of Zika virus with microcephaly, suggest its association with adverse pregnancy outcomes, and demonstrate evidence of Zika virus replication and persistence in fetal brain and placenta. This article highlights the value of tissue analysis to expand opportunities to diagnose Zika virus congenital and pregnancy-associated infections and to enhance the understanding of mechanism of Zika virus intrauterine transmission and pathogenesis. In addition, the tissue-based RT-PCRs extend the time frame for Zika virus detection and particularly help to establish a diagnosis retrospectively, enabling pregnant women and their healthcare providers to identify the cause of severe microcephaly or fetal loss.


This coinfection strain was determined to be of the Asian genotype and contains a novel insertion in the  Togaviridae and was first isolated in Tanzania in 1952  (-Chikungunya virus (CHIKV) is an alphavirus in the family of  in 1952 . Due to  in 1952 . CHIKV is (DENV) . Due to s (DENV) . Althougs (DENV) -12, to din vitro limiting-dilution–plaque purification in Vero cells. The viral RNA was extracted from the virus supernatant using GENEzol reagent (Geneaid), and the cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase and random primers. Second-strand synthesis was performed using the NEB 2nd-strand synthesis kit . The purified double-stranded DNA was subsequently prepped using Nextera XT  and sequenced on the MiSeq (2 × 150 bp setting) located at the Monash University Malaysia Genomics Facility, Selangor, Malaysia. The draft genome was assembled using IDBA_UD , containing two open reading frames (ORFs) coding for the typical structural and nonstructural proteins.. Intriguingly, a unique amino acid insertion in the nsP3 gene was observed in this isolate in positions 376 to 451. This insertion consists of a duplicated sequence of part of the N-terminal domain of nsP3. Deletions of seven amino acids at this similar position starting from amino acid (aa) 376 were observed initially in a few of the Malaysian isolates, later in Indonesian isolates, and isolates currently circulating in the Western Hemisphere  with strains from the Asian genotype with close similarity (99%) strain MY/06/37348 (accession no. FN295483.3) and MY/06/37350 (accession no. FN295484.2) isolated from Malaysiamisphere , 15. nsPKX168429.The complete genome of coinfected chikungunya virus, Asian genotype, has been deposited in GenBank under the accession no.


Aging societies represent a major challenge for health care systems all over the world. As older people tend to be more physically inactive, economic costs of inactivity are likely to increase notably. The present study aims to investigate this relationship between an aging society and economic costs of inactivity using the example of Germany.Using data from the German Socio-Economic Panel, this study applied the comparative risk assessment method developed by the WHO to estimate the direct costs of inactivity for the period 2001–2013 differentiated by gender-specific age-groups . Based on population statistics predicting the aging of the German population for the years 2014–2060, this research projects the development of future costs of inactivity and potential effects of interventions promoting physical activity among the German population.The results reveal an increase in the level of physical activity during the observed period (2001–2013) which compensated the negative effect of aging and resulted in a decline of inactivity costs. The projections for the years 2014–2060 indicate a constant increase in direct per capita costs until 2060 because of an aging society. Scenarios indicating how a short-term reduction of physical inactivity impacts costs of inactivity reveal the crucial role of the oldest age-group in this context.The findings indicate that the aging of the German population demands further actions and initiatives to promote physical activity, especially for the oldest age-group. Demographic trends projected over the next decades indicate a considerable aging of the world’s population . NotableAs a consequence, governments developed strategies to counteract the alarming development for certain clinical conditions. To that end, physical activity represents an effective mechanism. Research has identified particularly older people to be physically inactive . While tWith regard to health care costs, increasing physical activity rates is important because inactivity was identified to be associated with considerable costs for public health care systems in general –21. AcroThe overarching objective of this research is to examine how an aging population affects the economic costs of inactivity and how physical activity can assist governments in compensating rising health care costs of aging societies. The research context for this study is Germany – one of the Western countries which will be affected by an aging population in the next decades . The preThe contribution of this study to the existing literature is three-fold. First, it is one of the first studies estimating the development of direct costs of inactivity over a longer period of time. Existing studies have only estimated the costs for one specific year. Second, it provides projections about the development of future costs of inactivity in the context of an aging society and thirdly, this study aims to quantify potential economic savings which could be generated through short-term physical activity promotion and resulting reduction in inactivity levels, respectively. These projections are important for policy development in light of an aging society.The first purpose, the estimation of inactivity costs for the period 2001–2013, requires information about the annual direct health costs and annual inactivity rates. The Federal Statistical Office  providedInformation about levels of physical inactivity in Germany stems from the German Socio-Economic Panel (GSOEP), a panel survey with annual measurements from 1984 to 2013 . This sun = 166,309 observations from n = 41,991 individuals. More than half of the respondents were female (52.4%). The average age was 49.0 years with a range of 16–103 years. Certain population subgroups  are intentionally overrepresented in the survey. Consequently, specific sample weights provided in the dataset were used to ensure its representativeness of the overall German population [Depending on the respective wave, respondents could answer on a four- or five-point scale. Lechner  stated tpulation .The second and third purpose require predictions about future demographic changes of the German population. The Federal Statistical Office  has publIdentification of diseases related to physical inactivity including their relative risksIdentification of the annual direct costs of each diseaseDetermination of the prevalence of physical inactivity in Germany (2001–2013)Calculation of the population attributable fractions (PAF) for each diseaseMultiplying the PAFs with the annual direct costs of each disease to determine annual direct cost of physical inactivity.This section presents the analysis strategy in the order of purposes. For the first purpose, the prevalence based comparative risk assessment method developed by the World Health Organization (WHO)  was applRegarding the first step, the WHO  stated tTable For the third step, inactivity rates of the German population were estimated Table  using thIn a fourth step, by using the rates of inactivity and the RR of each disease, PAFs for each disease were calculated. A PAF identifies the proportion of a disease which can be associated with a certain risk factor . It is estimated with the following equation:Considering the second purpose, the direct per capita inactivity costs of each disease for every gender-specific age-group from the year 2013 were multiplied by projected age- and gender-specific population numbers for the years 2014–2060. The annual total direct costs per disease were then aggregated to estimate total direct costs of physical inactivity for every year. This method has been previously applied to forecast the impact of aging on health costs .The third research purpose builds on the projections of the second purpose, but considers two fictive scenarios predicting how future short-term actions to promote physical activity could impact projected direct costs of inactivity. The first scenario assumes that the inactivity rates in all age-groups could be reduced by five percentage points until 2020. The authors decided to use five percentage points because this value reflects the observed negative compound annual growth rate of physical inactivity of 3.6% for the period 2001–2013. Moreover, this figure has already been used in previous studies to estimate the impact of fictive physical activity reduction targets , 36. TheSince all three purposes are based on calculations using numerous input factors, this study conducted sensitivity analyses considering variations among those determining factors. Therefore, in line with Ding et al. , an extrThe results related to the first purpose of providing evidence from the past are visualized in Fig. During the 13-year observation period, the costs of inactivity have declined almost constantly. In 2013, the overall direct cost were reduced by 24.7% to €2.11 billion and €30.10 per capita, respectively, which represents a negative compound annual growth rate of 2.3%. Consequently, the proportion of total direct health care costs was reduced to 0.9%. For 2013, the best-case scenario indicated costs of €1.26 billion and the worst-case scenario costs of €3.19 billion.Figure For the second purpose of projecting the effects of aging on the costs of inactivity for the years 2014–2060, the predictions of the population development provided by the Federal Statistical Office  indicateFigure Figure The results of the third purpose analyzing two scenarios with fictive short-term targets of reducing physical inactivity are displayed in Table This study investigated the influence of physical inactivity – a major risk factor for numerous diseases – on the development of health care costs in Germany in the light of an aging population. It had three different research purposes focusing on (1) evidence from the past, (2) the prediction of future costs of inactivity, and (3) the effect of future physical activity promotion. Regarding evidence from the past, 1.3% of total direct health costs could be attributed to physical inactivity in 2001. This finding is in line with previous research from several countries, including the UK, Canada, and the United States , 39, 41 The costs of inactivity have significantly declined over the 13-year timespan. This development was driven by an overall decrease in inactivity rates in Germany. At least two explanations can be advanced for this finding. First, the results may indicate a rising awareness of the German population for the individual benefits of physical activity , potentiGerman baby boomers [Turning to the second purpose, the projected progression of future total costs of inactivity for the period 2014–2060 shows a plateau to be reached in 2039. One explanation for the following almost constant annual direct costs might be that at the end of the 2030s most of the  boomers  – born bThe third purpose of this study was to estimate the effect of those potential initiatives to reduce physical inactivity on direct costs of inactivity. The results stress the economic importance of promoting physical activity in particular among the elderly as a decrease in physical inactivity by 5 percentage points only in the oldest age-group of 65+ years can generate 73.6% of the costs savings if physical inactivity is reduced by 5 percentage points among all age-groups.Concerning policy development, it is important to consider that policies aimed at reducing physical inactivity bear different effects for socio-demographic groups. For example, Humphreys and Ruseski  revealedThe results of this study add to the existing literature in at least three ways. First, it is one of the first to analyze direct costs of inactivity over a longer period of time using panel data, enabling insights how costs are influenced by changing physical activity patterns of a population and demographic changes . Previous research has only used cross-sectional data and estimated the economic costs of inactivity for one year , 39, 41.The findings come with implications for policy makers and public health organizations. Overall the results indicate that the promotion of physical activity can be used as an important non-pharmaceutical action to reduce public health care costs considerably. Moreover, especially increasing physical activity rates among the elderly can assist governments in compensating the increasing demand for health care resulting from an aging population. In addition, the findings outline the urgency of short-term actions because a – well feasible – reduction of physical inactivity within the next five years could lead to long-term economic benefits for the national health care system. Since approximately 75% of cost savings can be attributed to the oldest age-group of 65 + years, initiatives and campaigns promoting physical activity should particularly target this age-group. Considering that costs occur for both designing and launching such campaigns and initiatives and their efficiency can differ significantly between age-groups , promotiThis study has some limitations which could guide future research. First, it is necessary to note that the population projections made by the Federal Statistical Office  only con