Recently discovered tick-borne phleboviruses have been associated with severe disease and death among persons in Asia and the United States. We report the discovery of a novel tick phlebovirus in Tasmania State, Australia, that is closely related to those zoonotic viruses found in Asia and North America. Bunyaviridae can infect animals and plants  , indicating that the men were independently infected with the same virus. This new virus, named Heartland virus (HRTV), was most closely related to, but clearly distinct from, the SFTSV detected in China  colony on Albatross Island, a small island in the Hunter Island Group in northwestern Tasmania, Australia. The disease in the albatrosses was characterized by weight loss and death. Serum samples and ticks (Ixodes eudyptidis) from healthy and affected birds were sent to the CSIRO  Australian Animal Health Laboratory in Geelong, Victoria, Australia, for diagnostic investigation. Several laboratory tests were conducted to detect potential viral pathogens involved in the disease event.In 2002, a disease outbreak occurred in a shy albatross (The ELISA or hemagglutination inhibition assay was used to test serum samples (N = 38) for antibodies against avian influenza virus, infectious bursal disease virus, fowlpox virus, and Newcastle disease virus. Neutralizing antibodies for Newcastle disease virus (titer of 64) were detected in 1 serum sample; otherwise, no antibodies against any of these suspected agents were detected.Pooled tick homogenates (N = 5) were cultured on chicken embryos, chicken embryo fibroblast and skin cells, and Vero cells. No virus was detected in chicken embryos or in any of the chicken cell lines used. In contrast, cytopathic effect was observed on the third passage in Vero cell cultures inoculated with 2 tick pools .Electron microscopy examination revealed morphologically similar viral particles from both cultures; the morphologic features resembled those of bunyaviruses . HoweverThe investigation was put on hold until 2011 when the Australian Animal Health Laboratory established its next-generation sequencing capability. RNA extracted from partially purified virus grown in Vero cells was used for random PCR amplification. The resulting products were used for library preparation and subsequently sequenced by using the 454 GS FLX system  according to standard protocols (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis at the nucleotide level showed no sequence identity to any of the known viruses in GenBank (data not shown). However, a protein-based BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search yielded 10 contigs showing protein sequence identity to SFTSV and some degree of sequence identity to several other members of the family Bunyaviridae. The virus was tentatively named Hunter Island Group virus (HIGV) after the location where the tick samples were obtained.BLASTN  that were responsible for severe disease and death in humans in 4 separate countries in Asia and North America. However, with the current data alone, the particular disease event in the shy albatrosses could not be attributed to HIGV.The findings from this study demonstrate the key role that a vigilant pathogen investigation has in any diagnostic assessment. The study findings also suggests that zoonotic phleboviruses genetically related to SFTSV, HRTV, and HIGV may be widely distributed in different parts of the world and that heightened international surveillance is needed to fully understand and appreciate the public health risk from these emerging viruses.


To the Editor: Parainfluenza virus 5 (PIV5), family Paramyxoviridae, genus Rubulavirus, was previously known as simian virus 5 because of its discovery in primary monkey kidney cells in 1954  in Baicheng City, Jilin Province, China. Initial clinical signs included secretion of clear nasal mucus, anorexia, sluggishness, and loss of bodyweight. After 10–20 d, ≈10% of the sick calves died of dyspnea and interstitial pneumonia. Farmers observed that 80%–90% of calves in the affected farms demonstrated clinical signs, but most recovered. All attempts of local veterinarians to treat the animals with various chemical compounds and antimicrobial drugs failed. The disease persists throughout the year but occurs mainly during spring (from February through March), which has resulted in substantial economic losses in the cattle industry.To identify the causative agent of the disease, we tested 15 lung samples from calves that had died and 10 lung samples from healthy calves that were slaughtered for serum products . The samples were homogenized in phosphate-buffered saline and analyzed by using electron microscopy. Paramyxovirus-like particles were identified in the tissues of sick calves. Reverse transcription PCR with the generic primers for the paramyxovirus polymerase gene was performed . After virus adsorption for 1 h at 37°C, the cell monolayer was rinsed with phosphate-buffered saline (pH 7.4) and then incubated in Dulbecco minimal essential medium/2% newborn calf serum at 37°C in a 5% CO2- humidified incubator. The infected cells were serially passaged every 3 days at 37°C and detected by using monoclonal antibody against SV5  by indirect fluorescent antibody test  was observed between PIV5-BC14 and porcine PIV, particularly with the SER virus isolate. This observation was confirmed after construction of a phylogenetic tree based on the 15 available nucleotide sequences of the full-length genomes . The anaAlthough the pathogenic role of PIV5 infections in cattle remains unknown, no PIV5 RNA was found in any apparently healthy cattle from the same farm. This result suggests a strong relationship between the identified virus and the disease.


To review the state of knowledge about diagnostic testing for Zika virus infection and identify areas of research needed to address the current gaps in knowledge.We made a non-systematic review of the published literature about Zika virus and supplemented this with information from commercial diagnostic test kits and personal communications with researchers in European preparedness networks. The review covered current knowledge about the geographical spread, pathogen characteristics, life cycle and infection kinetics of the virus. The available molecular and serological tests and biosafety issues are described and discussed in the context of the current outbreak strain.We identified the following areas of research to address current knowledge gaps: (i) an urgent assessment of the laboratory capacity and capability of countries to detect Zika virus; (ii) rapid and extensive field validation of the available molecular and serological tests in areas with and without Zika virus transmission, with a focus on pregnant women; (iii) monitoring the genomic diversity of circulating Zika virus strains; (iv) prospective studies into the virus infection kinetics, focusing on diagnostic sampling ; and (v) developing external quality assessments for molecular and serological testing, including differential diagnosis for similar viruses and symptom clusters. The availability of reagents for diagnostic development  needs to be facilitated.An international laboratory response is needed, including preparation of protocols for prospective studies to address the most pressing information needs. On 1 February 2016, the World Health Organization (WHO) declared that the recent cluster of microcephaly cases and other neurological disorders reported in the Americas, where an outbreak of Zika virus infection is ongoing, constitutes a public health emergency of international concern.Flaviviridae and genus Flavivirus. Flaviviruses are positive-sense ribonucleic acid (RNA) viruses, with a genome of approximately 11 kilobases. Virions are produced as spherical particles, 40–60 nm in diameter.,Zika virus is a mosquito-borne virus related to yellow fever, dengue, West Nile, Japanese encephalitis and tick-borne encephalitis viruses which all belong to the virus family ,––Zika virus infection is often asymptomatic; around 80% of patients showed few or no clinical symptoms during the outbreaks in Yap Island and French Polynesia.,,A major concern is the possible association of Zika virus infection with Guillain–Barré syndrome – a neurological disorder – and with microcephaly and other neurological manifestations in newborns of infected mothers. These complications have been noticed in the current epidemic region and were identified retrospectively in the French Polynesia outbreak.The Zika virus outbreak is also likely to increase the number of cases exported from epidemic areas by travellers . The curWe made a non-systematic review to present the essential background information and current gaps in knowledge about diagnostic testing for Zika virus infection in humans. We made a literature search of the PubMed database up to 21 January 2016, using the search terms “Zika virus”, “ZIKV” or “Zika”, with no date or language restriction. This was supplemented by information obtained from commercially available diagnostic test kits and from personal communications with researchers in European preparedness networks. We identified areas of research and actions needed to address the identified gaps in international laboratory preparedness for an adequate response to the current outbreak.Serological, epidemiological and entomological studies have reported the circulation of the virus in tropical areas of western Africa, central Africa and in Asia.,Zika virus belongs to the Spondweni virus serogroup of mosquito-borne flaviviruses . PhylogeAedes mosquitoes; Ae. aegypti is the only species for which transmission outside Africa has been confirmed. In the 2007 outbreak on Yap Island, Ae. hensilii mosquitoes were implicated as the vector, but this could never be confirmed by virus detection. The virus has been isolated and/or detected by reversed transcriptase (RT) polymerase chain reaction (PCR) assay from the following species in the field in Africa: Ae. africanus, Ae. aegypti, Ae. albopictus, Ae. apicoargenteus, Ae. luteocephalus, Ae. vitattus, Ae. taylori, Ae. dalzieli, Ae. hirsutus, Ae. metallicus, Ae. unilinaetus, Ae. opok and Ae. furcifer.,Mansonia uniformis, Culex perfuscus and Anopheles coustani. Ae. albopictus has shown competence in Zika virus dissemination in laboratory studies but has never been implicated in Zika virus epidemiology in the field outside of Africa.,Zika virus is transmitted by Additional modes of transmission have been identified. Perinatal transmission most probably occurs by transplacental transmission or during delivery by an infected mother.Knowledge of the Zika virus infection kinetics is essential to determine the optimal strategy for diagnosis. This is hindered by the fact that only a few diagnosed cases of human Zika virus infection, all of Asian lineage, are described in the literature.–,,,,Data from the French Polynesia outbreak described viraemia that was of low intensity and short duration.Typically for flaviviruses, immunoglobulin (Ig) M antibodies develop within a few days after onset of illness and can generally be detected for up to 3 months. IgG antibodies develop within days after IgM and can be detected for months to years. Cases have been described with persistence of IgM antibodies for longer periods, which complicates accurate diagnostic testing.,,Immune response from Zika virus infection has only been described in 11 patients during the Zika virus outbreak on Yap Island. IgM was detected as soon as 3 days after the onset of symptoms. IgG appeared after day 10 in a patient with no history of previous flavivirus infections.Specific attention should be given in prospective studies to determine the Zika virus immune responses in pregnant women, since antibody responses during pregnancy may be different from those in non-pregnant women.To run molecular tests in an outbreak setting, laboratories need knowledge and experience of quality control and validation, access to rapid supply chains for reagents and plastics, and the capability to increase the throughput of testing. To speed up testing, experience with automated equipment for nucleic acid extraction and RT–PCR assay are essential.In serological assays, extensive cross-reactivity between virus genus members or geographical overlap with other pathogens causing overlapping syndromes might occur. In these cases, pan-genus and syndromic serum panel tests and antigens need to be available and confirmatory techniques such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay and virus neutralizing testing are required.http://www.who.int/bulletin/volumes/94/8/15-171207).Molecular diagnosis of Zika virus can be done on different types of body fluids: whole blood, serum, EDTA (ethylenediaminetetraacetic acid) plasma, saliva and urine. Urine and saliva should be considered together with blood and/or serum in the algorithm of Zika virus genome detection using molecular techniques. The reliance on the use of molecular diagnostics to rule out infection requires careful consideration, as the experience of clinicians and diagnostic laboratories is necessarily limited for emerging diseases. Several non-commercial RT–PCR tests for Zika virus have been described in the literature, but few provide validation using the most recent viral strains and fully documented clinical specimens. In this article, we only discuss the 12 RT–PCR assays that have resulted in the detection of viral RNA in at least one human case of Zika virus infection, as described in a peer-reviewed article indexed in PubMed or in a personal communication . We have,,,Various real-time and conventional RT–PCR assays specific for Zika virus have been described .19,43,51Other researchers have described an assay using a locked nucleic acid probe in a real-time RT–PCR assay for detection of Zika virus in human serum ,52 althoin silico assessment of their fit with the current Zika virus. Commercial kits on the market include: RealStar® Zika virus RT–PCR kit 1.0 , Genesig® Zika virus Advanced kit , MyBioSource Zika real time RT–PCR kit , Genekam Zika virus PCR  and FTD Zika virus RT–PCR kit . Manufacturers should be encouraged to put detailed information about their primers and probes into the public domain, so that the performance of these can be evaluated continuously in the context of our evolving knowledge about the genomic diversity of Zika virus during the current outbreak.Commercial RT–PCR assays for Zika virus are rapidly becoming available. However, until now the tests are for research purposes only. The primers and probe sequences in commercial kits are not usually publicly available, which precludes ,–Flavivirus) RT–PCR assays have been used in combination with sequencing to detect human Zika virus cases  were used to map antibody responses in 11 patients from the Yap Island outbreak.To the authors’ knowledge three commercial tests are on the market at the time of writing this article or will be available soon. The Zika virus IgG and IgM detection kits of MyBioSource Inc.  use a double-antigen sandwich ELISA. No information on the type of antigens used or on the test specificity and sensitivity is given by the manufacturer.–Zika virus is classified as a biosafety level 2 pathogen in the European Union (with the exception of the United Kingdom) and the USA. There are no inactivation data available that are specific for Zika virus. However, flaviviruses are typically inactivated by temperatures above 56 °C for at least 30 minutes, in solutions of pH ≤ 6, by ultraviolet light and by gamma-radiation and are known to be susceptible to disinfectants such as 1% sodium hypochlorite, 2% glutaraldehyde, 70% ethanol, 3–6% hydrogen peroxide and 3–8% formaldehyde.During the initial steps of molecular detection methods phenol guanidine isothiocyanate or chaotropic salts are added to the samples to extract the RNA. These reagents inactivate flaviviruses, and therefore the diagnostic procedure could continue in a laboratory with standard safety levels after the addition of these reagents.Although Zika virus is only a level 2 pathogen, laboratories should assess the additional risks for laboratory personnel who are pregnant, especially when the virus is cultured .Box 1• The following areas of research are suggested to address the knowledge gaps identified: – Urgent assessment of the laboratory capacity and capability of countries to detect Zika virus.– Conducting rapid and extensive field validation of the available molecular and serological tests in areas with Zika virus transmission and in areas unaffected by transmission but receiving returning travellers. Special focus should be on pregnant women.– Monitoring the genomic diversity of circulating Zika virus strains to allow verification against operational molecular tests to ensure continuous sensitivity of testing.– Conducting prospective studies into the Zika virus infection kinetics in the general population and in pregnant women. This should focus on determining the ideal types of diagnostic samples , combinations and times of collection (stage of illness).– Developing external quality assessments for both molecular and serological testing. Besides Zika-virus-positive samples, these panels should include: (i) a cross-reactive panel, consisting of viruses or antibodies to closely related viruses  and (ii) a syndromic panel with pathogens or antibodies to diseases with a clinical manifestation similar to Zika virus . External quality assessments for molecular testing should address different types of diagnostic samples.• The availability of reagents for diagnostic development  needs to be facilitated. This could be achieved through initiatives such as the European Virus Archive• An international laboratory response is needed, which would include preparation of protocols for prospective studies to address the most pressing information needs.


Human bocavirus (HBoV) was identified as the second human parvovirus with pathogenic potential in 2005 in respiratory samples from children suffering from viral respiratory infections of unknown etiology. Since its first description, a large number of clinical studies have been performed that address the clinical significance of HBoV detection and the molecular biology of the virus. This review summarizes the most important steps taken in HBoV research to date and addresses open questions that need to be answered in the future to provide a better understanding of the role of a virus that is difficult to grow in cell culture and is suspected to be a pathogen, although it has not yet fulfilled Koch’s postulates. For a long time, the number of respiratory infections with clinical symptoms caused by respiratory viruses had no detectable causative agent. With the discovery of human metapneumovirus in 2001 by Bernadette van den Hoogen and her colleagues [Since the discovery of HBoV, numerous studies related to the epidemiology, pathogenesis, and replication of this newly detected virus have been performed, and they have provided novel insights into viral infection of the respiratory tract and gastrointestinal diseases.Parvoviridae, subfamily Parvovirinae, and genus Bocavirus. Currently, four subtypes have been identified, and of these, HBoV1 is most frequently detected in clinical samples of the respiratory tract. The remaining isolates are more frequently associated with gastrointestinal infections and symptoms [Based on sequence analyses of the 17 isolates identified by Allander and coworkers, HBoV was grouped into the family symptoms ,4,5,6.Parvoviridae family, have been identified as linker sequences in episomal genomes or replication intermediates in HBoV1 [The HBoV genome length ranges from 5,217 to 5,299 nucleotides  plus thein HBoV1  and havein HBoV1 , the latRecently, Zhao and colleagues showed that the genome of HBoV subtype 2 is circular . This ciTo date, the genome replication of HBoV has been assumed to occur in the same manner as the genome replication of animal parvovirus, that is, via a rolling hairpin mechanism, which would generate head-to-head or tail-to-tail intermediates, but the recently detected head-to-tail sequences do not fit with this replication model ,8. A novIt appears likely that HBoV either persists or uses additional or alternative mechanisms of replication, which remains a matter of speculation.cis and in trans [An alternative replication model could be a helper virus-dependent initiation of rolling circle replication. In a recent case report (below), we identified herpesvirus 6 co-infection in an HBoV-infected patient. Surprisingly, during cidofovir therapy, HHV-6 viremia decreased, and HBoV was eliminated, even though the patient had an underlying immunodeficiency and could not produce neutralizing antibodies . Considein trans , and givin trans , this reBocavirus genus. The viral genome contains at least three open reading frames (ORFs). Although it has not been experimentally verified for all encoded genes, it is widely assumed and likely that the proteins encoded by the HBoV genome are similar in function to the proteins of better characterized members of the Bocavirus genus. Thus, the first ORF at the 5' end likely encodes a nonstructural protein known as NS1, whereas the subsequent ORF likely encodes a second non-structural protein, NP1, that is believed to be unique to bocaviruses. The third ORF encodes the VP1 and VP2 proteins.The viral transcriptome has been analyzed in 2 studies, which revealed that RNAs for the proteins NS1, NP1, UP1/NP1, VP1/VP2, UP2/VP1/VP2, a putative ORFx protein, and an NS1-70 protein ,19 are mThere is currently little information on the interactions of HBoV proteins with each other and with cellular proteins, primarily because secondary reagents, such as HBoV protein-specific antibodies and a versatile cell culture system, are not available. These materials are necessary for the investigation of such interactions. The novel reverse genetics system published by Huang and colleagues could be a useful tool for the analysis of the functions of HBoV proteins.Overexpressed VP2 protein is able to form capsid-like structures that strongly resemble viral particles . VP2 parHBoV is a major pathogen detected in respiratory and gastrointestinal infections. To date, four subtypes have been identified, and they are found worldwide, without any regional, geographic, or border restrictions. Following its initial discovery in Sweden, HBoV has been detected all over Europe ,30,31,32The four distinct subtypes have been named HBoV1–4. Subtype 1 is mainly associated with respiratory diseases, but can also be found in stool samples from patients suffering from diarrhea. The prevalence in symptomatic patients is approximately 1.5–16% for HBoV-1 ,52 21–26The seroprevalence of HBoV is strongly dependent on the age of the investigated patient cohort and ranges from ~40% in children between 18 and 23 months of age up to virtually 100% in children older than two years, with an average of 76.6% in children and 96% in adults ,56. The i.e., infections that are clinically relevant, require hospitalization, and receive a proper laboratory diagnosis) frequently represent co-infections with up to six different pathogens in a single patient [Recent clinical studies on respiratory infections that used novel multiplexing assays have shown that more severe infections  has consequently classified this virus as a biosafety level 2 agent.Due to the lack of a cell culture system, the diagnosis of HBoV infection is exclusively based on molecular detection methods. Most laboratories currently use in-house PCR and real-time PCR assays targeting the NP-1, NS-1 or VP1/2 gene , but othTo correctly diagnose an HBoV infection, it is necessary to screen clinical samples from the respiratory tract or stool samples  and a corresponding serum sample ,69,70,72Although it was discovered several years ago, there are still more questions than answers related to human bocavirus. Despite an increasing and overwhelming amount of evidence that the virus is in fact a pathogen, Koch’s modified postulates have not yet been fulfilled for HBoV, primarily because a versatile cell culture system and an animal model are not available. Moreover, the virus is frequently detected as a co-pathogen, primarily because with the discovery of HBoV, a new era of clinical studies of respiratory infections has begun. In these studies, patients are screened for virtually all viruses known at the time the studies are performed. Currently, the primary challenge of the HBoV community is to develop a cell culture system or an animal model in which the virus can be propagated. The most recently published cell culture system using CuFi and NuLi cells could be such a model.These tools are a prerequisite for the search for answers to open questions, including questions regarding effective antivirals and disinfectants and the molecular pathogenesis of HBoV. These tools will also help to determine whether HBoV is in fact a serious pathogen and not simply part of the harmless microbiome of its hosts.Based on the currently available data, until HBoV can be proven to be a harmless bystander, I recommend that HBoV be treated as a pathogen, and thus, an inverse presumption of innocence should apply for HBoV.If one tries to follow the history of virus discoveries during the last decade and has performed clinical studies in the field of respiratory infections, it is obvious that with the increasing number of identified pathogens, the range of diagnostic tools required in a clinical study has also increased. Thus, the more known viruses there are, the more diagnostic assays are demanded by investigators, clinicians, and peer reviewers of the manuscripts that describe those studies. It thus remains unknown whether HBoV is in fact a pathogen or whether the co-detection rate was high because clinical studies on HBoV infections were among the first generation of studies in which the presence of virtually all respiratory pathogens was required to be assessed.


The complete genome of a foot-and-mouth disease type O virus originating from an epidemic in Greece in 1994 is reported. This virus belongs to the Middle East-South Asia topotype. Aphthovirus within the family Picornaviridae and causes FMD in cloven-hooved animals. FMD virus has a positive-sense single-stranded RNA genome of approximately 8.3 kb, which contains a single open reading frame (ORF) encoding 4 structural and 10 nonstructural proteins (Foot-and-mouth disease (FMD) virus belongs to the genus The 1994 FMD virus (FMDV) epidemic in Greece began with high morbidity in sheep. After approximately 1 month there was a sharp decline of clinical cases within sheep flocks, while morbidity within cattle and pig populations remained stable. There is evidence to suggest that FDMV cannot maintain itself within sheep populations for long periods of time , and thede novo assembler program IDBA UD (version 1.1.1) (https://github.com/josephhughes/btctools). The total length of the genome recovered was 8,161 nucleotides with a mean coverage depth of 1,634 across the genome; we were not able to determine the sequence or length of the poly(C) tract or 7 adjacent downstream nucleotides. Comparison of O/GRE/23/94 with nine sequences belonging to the Middle East-South Asia topotype (–O/GRE/23/94 was obtained from the World Reference Laboratory for FMD (WRLFMD) collection  and was isolated, using primary calf thyroid cells, from ovine epithelium collected on 4 August 1994 in Xanthi during the peak of the Greek FMD epidemic (n 1.1.1) , with ann 1.1.1)  was usedn 1.1.1)  was usedn 1.1.1) , coveragtopotype  (AJ53913topotype –13. The opotype –.KU726614.The nucleotide sequence for FMDV O/GRE/23/94 has been deposited in GenBank under accession number


Haptolina (formerly Chrysochromulina) ericina. CeV-01B and its closest relative Phaeocystis globosa virus define an emerging subclade of the Megaviridae family with smaller genomes and particles than the originally described giant Mimiviridae infecting Acanthamoeba.We report the complete genome sequence of CeV-01B, a large double-stranded DNA virus infecting the unicellular marine phytoplankton  Haptolina (formerly Chrysochromulina) ericina is a phytoplankton species with a worldwide distribution. It most commonly occurs in low numbers but has occasionally been observed to form blooms (Chlorella spp. (–Emiliania huxleyi (Coccolithophyceae) (Phaeocystis globosa (Prymnesiophyceae) (Aureococcus anophagefferens (Pelagophyceae)  , Phaeocyphyceae) , and Aurphyceae) .Haptolina ericina virus CeV-01B was isolated from Norwegian coastal waters in 1998 (k = 97). Two scaffolds, with sizes of 67 kb and 410 kb were initially obtained. The scaffolder SSPACE , of which 163 (59.5%) were most similar to their PgV homolog and an additional 40 had their closest homologue in other Megaviridae. These best matches in PgV include usual phylogenetic markers such as the DNA polymerase B , the major capsid protein , as well as two enzymes characteristic of Megaviridae: the mismatch DNA repair enzyme MutS7 , using GeneMark , and 12 me MutS7    , progresbfamily) , 13, 21.bfamily)  with PgVHaptolina ericina virus CeV-01B is available in Genbank under accession number KT820662.The completely annotated genomic sequence of


These centers could lead to a decline in cases, even if virus containment is imperfect. In some parts of western Africa, Ebola treatment centers (ETCs) have reached capacity. Unless capacity is rapidly scaled up, the chance to avoid a generalized Ebola epidemic will soon diminish. The World Health Organization and partners are considering additional Ebola patient care options, including community care centers (CCCs), small, lightly staffed units that could be used to isolate patients outside the home and get them into care sooner than otherwise possible. Using a transmission model, we evaluated the benefits and risks of introducing CCCs into Sierra Leone’s Western Area, where most ETCs are at capacity. We found that use of CCCs could lead to a decline in cases, even if virus transmission occurs between CCC patients and the community. However, to prevent CCC amplification of the epidemic, the risk of Ebola virus–negative persons being exposed to virus within CCCs would have to be offset by a reduction in community transmission resulting from CCC use. The current epidemic of Ebola virus disease in western Africa has resulted in thousands of cases during 2014 , which would represent a possible change in operational approach . Because precise medical and operational details of CCCs are still under discussion, we focused on the implications of CCC introduction under a set of general assumptions. We modeled Ebola transmission by using a modified susceptible-exposed-infectious-resolved framework . In the model, Ebola virus–positive and –negative patients took an average of 4.6 days ; we did not assume any transmission of virus from ETC patients. There was also a chance that Ebola virus–negative patients would be exposed to Ebola virus while waiting for test results. We assumed that 50% of symptomatic patients who attended CCCs/ETCs were Ebola virus–positive; on the basis of the number of Ebola virus disease cases and noncases reported in Sierra Leone, this percentage is plausible  the probability that Ebola virus–negative patients would be exposed to Ebola virus while waiting for test results in CCCs and 2) the reduction in virus transmission from infectious patients to the community as a result of the patient being isolated in a CCC. If the CCC model had a 100% reduction in transmission and 0% chance that Ebola virus–negative patients would be exposed virus, it was equivalent to the ETC set-up in the model, except that there would be a reduced time from symptom onset to CCC attendance.We first considered the potential level of infection in the community during December 2014 based on our estimates for Western Area. With 259 ETC beds available , panel ATo assess what reduction in transmission and in risk of Ebola virus–negative patient exposure to virus would be required for 500 CCC beds to be beneficial, we varied 2 key parameters and, after 30 days, compared model outputs with those for the baseline scenario , panel Ahttps://drive.google.com/file/d/0B_BzCqSK1DZaYnRoeWtHOTU2TVk/). The distribution for the group opinion for reduction in transmission while in a CCC had a median of 63% (IQR 41%–81%). The distribution for the probability of exposure had a median of 0.09 (IQR 0.01–0.36). When compared with model results, these estimates were within the region of parameter space in which CCCs would be beneficial .The CCC approach has not been fully tested in the field, so we conducted an elicitation of 6 expert opinions to obtain estimates for the median and interquartile range (IQR) for reduction in transmission as a result of patients being in CCCs and for the probability of exposing Ebola virus–negative patients to infectious patients . Even if Ebola patients had a 50% probability of infecting their sole caregiver, it meant they would, on average, generate 0.5 secondary cases while in a CCC. The relative reduction in cases as a result of being in a CCC would therefore be (1.4 − 0.5)/1.4 = 64%. If each case-patient generated an average of 0.25 cases while in a CCC, the expected reduction would be ≈80%.To confirm that 63% was a plausible value for reduction in transmission, we used the following theoretical argument. In the model, the basic reproduction number, To elucidate the potential benefits and risks of CCC introduction, we considered 2 specific examples. If CCCs reduced virus transmission from Ebola virus–infected patients to the community by 75% once the patient was admitted and if Ebola virus–negative patients have a 25% probability of exposure while waiting for test results, then the introduction of 500 CCC beds would slow virus transmission , panel BR, to <1). A larger number of beds would be required if the reduction in transmission was smaller (https://drive.google.com/file/d/0B_BzCqSK1DZaYnRoeWtHOTU2TVk/). The large number of infected persons on December 1, 2014, meant that the number of cases still rose in the model  if the time from symptom onset to CCC attendance is <3 days. Assuming that CCCs open in mid-December, ensuring epidemic turnover would require a large number of CCC beds . In addition to reducing the time from symptom onset to attendance at a treatment facility, a large number of CCCs would have the added benefit of reducing the time from symptom onset to admission because infected patients would not have to wait for ETC beds to become available.We used a transmission model to evaluate the potential effects of the introduction of Ebola CCCs in Western Area, Sierra Leone. Our results show that CCCs could reduce the number of Ebola virus disease cases in the community if 1) the probability for Ebola virus–negative patients being exposed to the virus is low and 2) there is reduction in virus transmission as a result of infected patients being in CCCs. The introduction of CCCs could potentially turn over the epidemic (i.e., reduce the reproduction number, Our analysis does have limitations. One of those limitations is that we used an illustrative scenario for Western Area based on current epidemiologic reports. Given uncertainty about the influence of factors such as changes in behavior (Furthermore, we assumed that infectiousness does not vary over the course of Ebola virus infection. However, if patients are most infectiousness during the final stages of infection (In summary, CCCs may offer a rapid, high-coverage complement to ETCs and, thus, hold considerable potential for bringing about a sizeable shift in the epidemic pattern in Sierra Leone. The UK government is therefore supporting such a combined intervention in the Sierra Leone (


KUN) caused an unprecedented epidemic of neurological disease in horses in southeast Australia, resulting in almost 1,000 cases and a 9% fatality rate. We investigated whether increased fitness of the virus in the primary vector, Culex annulirostris, and another potential vector, Culex australicus, contributed to the widespread nature of the outbreak.In 2011, a variant of West Nile virus Kunjin strain  observed were 68% for WNVKUN2011 on day 12 and 72% for WNVKUN2009 on day 14. On days 12 and 14 post-exposure, significantly more WNVKUN2011 than WNVKUN2009 was expectorated by infected mosquitoes. Infection, dissemination and transmission rates of the two strains were not significantly different in Culex australicus. However, transmission rates and the amount of virus expectorated were significantly lower in Cx. australicus than Cx. annulirostris.There were few significant differences between the two virus strains in the susceptibility of KUN2011 expectorated by infected mosquitoes may be an indication that this virus strain is transmitted more efficiently by Cx. annulirostris compared to other WNVKUN strains. Combined with other factors, such as a convergence of abundant mosquito and wading bird populations, and mammalian and avian feeding behaviour by Cx. annulirostris, this may have contributed to the scale of the 2011 equine epidemic.The higher amount of WNV KUN) is endemic in Australia, where it can cause a mild febrile illness and occasionally non-fatal encephalitis in humans and horses [Culex annulirostris, the primary mosquito vector [West Nile virus (WNV) is a mosquito-borne flavivirus that historically was responsible for outbreaks of acute encephalitis in Africa, Europe, Russia and the Middle East , but is d horses . The viro vector .KUN [KUN, designated WNVKUN2011, was subsequently shown to be the primary cause of neurological disease in horses [KUN is only rarely associated with equine disease [KUN from epidemic foci during 2011 [KUN in Victoria found less than 0.3% were IgM positive, providing little evidence of recent exposure amongst the human population [Between January and June 2011, a widespread outbreak of neurological disease attributed to arbovirus infection occurred amongst horses in southeastern Australia resulting in 982 reported equine cases, with an overall case fatality rate of 9% . In addiKUN . This stn horses ,6. This  disease ,7. Paradpulation , there wKUN was also reported in novel areas, such as east of the Great Dividing Range near the major coastal population centers of Sydney, Newcastle and Wollongong [Of note, other key epidemiological and biological features characterized this unique outbreak. La Niña-driven widespread rainfall and extensive flooding in southeastern Australia during the spring and summer of 2010–2011 . WNVKUN llongong ,6. This llongong .KUN2011 in mosquitoes, similar to what has been suggested for emergent strains of WNV in North America [KUN2011 strain in Cx. annulirostris and compare it to a recent strain of WNVKUN that has not been associated with recognized pathogenicity in horses or other vertebrates. Infection characteristics of the two virus strains were also evaluated in another potential vector, Culex australicus. This species is an indigenous member of the Culex pipiens group, a complex which contains major WNV vectors in North America and Europe [A key factor that may have led to the 2011 outbreak was increased fitness of WNV America ,14. To ed Europe ,16. It hd Europe , includiKUN2011 was isolated from the brain of a deceased horse from Boorowa , NSW. It had been passaged three times, once each in baby hamster kidney (BHK), African green monkey (Vero) and Aedes albopictus (C6/36) cells and the stock virus had a final titre of 108.9 tissue culture infectious dose (TCID)50/mL. The strain to which it was compared (WNVKUN2009) was isolated from Cx. annulirostris collected from Kununurra , Western Australia, in 2009. It had been passaged twice in C6/36 cells, once in porcine stable equine kidney (PSEK) cells before a final passage in C6/36 cells; the stock virus had a final titre of 108.7 TCID50/mL.The WNVCx. annulirostris and Cx. australicus were collected from a number of sites around Hexham Swamp , Fullerton Cove  and Kooragang Island  near Newcastle, NSW, using encephalitis virus surveillance (EVS) light traps [2 (approx. 1 kg dry ice). The mosquitoes were transported to the laboratory and placed in a 30 × 30 × 30 cm cage at 26°C, 75% humidity and 12:12 L:D. On the night following collection, mosquitoes were offered an anesthetized rat as a blood meal source. The use of animals was approved by the University of Sydney and Westmead Hospital Animal Ethics Committee . Two days later, 200 mL of water was added to the cage for oviposition. After two days, egg rafts were removed, placed on moist filter paper in a Petri dish and forwarded by overnight courier to Forensic and Scientific Services, Department of Health, Brisbane.Adult ht traps  baited w2O containing ~ 45 mg of brain-heart infusion powder. First and 2nd instar larvae were fed a 1:1 mixture of brewer’s yeast  and fish flakes , while 3rd and 4th instar larvae were fed on Hikari® Cichlid Staple pellets . Pupae were placed in 150 mL containers within a 30 × 30 × 30 cm cage and emerged adults were provided 15% honey water ad libitum. Prior to virus exposure, mosquitoes were sorted by species and 5–7 day old females placed into 900 mL gauze-covered containers.Upon receipt, eggs were hatched in 2.5 L of ddHCx. annulirostris were offered blood meals containing 10-fold dilutions of stock virus, whilst Cx. australicus was exposed to a single dose of virus only. To determine the titer of virus at the time of feeding, 100 μL samples of the pre- and post-feeding blood/virus suspensions were diluted in 900 μL of growth media with antibiotics and antimycotics  and supplemented with 3% fetal bovine serum (FBS), before being stored at −80°C.Mosquitoes were exposed for 2 hours to hanging drops of a blood/virus suspension containing stock virus, commercially available defibrinated sheep blood  and 1% sucrose . For tesFollowing feeding, mosquitoes were sorted and blood engorged mosquitoes placed in 900 mL gauze-covered containers. Containers were placed in sealed rigid plastic boxes within an environmental growth cabinet at 28°C and 12:12 L:D. Relative humidity was increased by placing a moist cotton wool pad within the plastic boxes and 10% sucrose was provided as a carbohydrate source.Cx. annulirostris fed the three lowest virus titers were killed via exposure to CO2 and placed individually in 2 mL vials containing 1 mL of GM +3% FBS and a 5 mm stainless steel ball. To compare virus replication and the length of the incubation period for the two viruses in Cx. annulirostris, on days 3, 5, 7, 10, 12 and 14, mosquitoes fed the highest virus titer were tested for their ability to transmit virus using the forced salivation method of Aitken [Cx. australicus were only tested for their ability to transmit at day 12. For transmission attempts, mosquitoes were anaesthetized with CO2 gas, their legs and wings removed and the proboscis of each mosquito inserted into a microcapillary tube containing 25 μL of GM +20% FBS. After approx. 30 min the contents of the tube were expelled into a 2 mL vial containing 500 μL of GM +3% FBS. The body remnants, and legs and wings were placed separately in 2 mL vials containing 1 mL of GM +3% FBS and a 5 mm stainless steel ball. Recovery of virus from the legs and wings is indicative of a disseminated infection, whereby the virus has escaped from the midgut and disseminated through the hemocoel [For the susceptibility trials, at day 14 post exposure, f Aitken . Due to hemocoel . The whoThe pre- and post-exposure blood/virus suspensions were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate containing confluent layers of C6/36 cells. Plates were incubated at 28°C for seven days, after which time they were fixed with PBS/20% acetone with 0.2% BSA and stored at −20°C.Mosquito homogenates were filtered through a 0.2 μm Supor® membrane filter . The body filtrate of the mosquitoes exposed to the three lowest virus titers, and the legs and wings filtrate, were inoculated in duplicate onto C6/36 cell monolayers within a 96-well microtitre plate. The filtrate of the remaining bodies and all saliva expectorates were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate containing confluent monolayers of C6/36 cells. Plates were incubated, fixed and stored as described above.KUN infection in all blood/virus mixtures and mosquito samples using the WNVKUN- specific monoclonal antibody, 10A1 [A fixed cell culture enzyme immunoassay (CCEIA) was used to detect WNVdy, 10A1 .50/mL. The susceptibility of Cx. annulirostris to infection with the two WNVKUN strains was calculated by probit analysis using PriProbit version 1.63 . Log-log models were assessed using the Pearson chi-square goodness-of-fit statistic and susceptibility to infection was expressed as ID50 ± 95% confidence intervals (CIs) and defined as the virus dose per mL at which 50% of Cx. annulirostris tested positive for WNVKUN infection in the CCEIA. Overlap of 95% CIs was used as a test of statistical significance.The titer of the blood/virus suspension, and the mosquito bodies and saliva expectorates was calculated using the method of Reed and Meunch  and exprCx. annulirostris and Cx. australicus exposed to WNVKUN2011 and WNVKUN2009 were compared using Fisher’s exact test [Cx. annulirostris and Cx. australicus exposed to WNVKUN2011 and WNVKUN2009 were analyzed for each day of exposure using t-tests [Infection, dissemination and transmission rates in act test . Differe t-tests .KUN2011 ranging from 104.4 to108.1 TCID50/mL, and 104.8 to 107.6 TCID50/mL for WNVKUN2009  TCID50 per mL  and 107.1  TCID50 per mL , respectively. Although there was no statistically significant difference in susceptibility to the virus strains, the overlap between CIs was very small.Mosquitoes were exposed to doses of WNVCulex annulirostris were exposed to virus titers of 108.1 and 107.6 TCID50/mL of WNVKUN2011 and WNVKUN2009, respectively. There was no significant difference (P > 0.05) in infection rates in Cx. annulirostris exposed to the two WNVKUN strains on any day post exposure, except at day 14, where significantly more mosquitoes were infected with WNVKUN2009 compared to WNVKUN2011  in the overall dissemination rate or the rate of dissemination in infected mosquitoes between Cx. annulirostris exposed to the two WNVKUN strains on any day post-exposure (Table P > 0.05) in the overall transmission rate or the transmission rate in mosquitoes with a disseminated infection on any day post-exposure.Cx. annulirostris infected with WNVKUN2011 were higher than those infected with WNVKUN2009 . Conversely, on day 14, WNVKUN2009-infected mosquitoes had significantly higher body titers than WNVKUN2011-infected mosquitoes (P < 0.001), which coincided with the significantly higher infection rate observed above. On days 10–14, when a greater proportion of mosquitoes was transmitting the virus, there was a higher amount of virus expectorated by WNVKUN2011-infected mosquitoes, with significant differences observed on days 12 and 14  in infection, dissemination and transmission rates in Cx. australicus 12 days after being exposed to virus titers of 108.1 and 107.6 TCID50/mL of WNVKUN2011 and WNVKUN2009, respectively (Table P > 0.05) from those observed for Cx. annulirostris. However, the overall transmission rate was significantly lower (P < 0.05) in Cx. australicus than Cx. annulirostris for both WNVKUN2011 and WNVKUN2009  for WNVKUN2011.There was no significant difference  than those exposed to WNV2009  than the body titers observed for both viruses in Cx. annulirostris. There was no significant difference (P > 0.05) between the amount of WNVKUN2011 or WNVKUN2009 expectorated by Cx. australicus  for WNVKUN2011.The body titers in 9 Figure A. They ws Figure B. HoweveKUN2011 were investigated to ascertain why there was such a dramatic impact from what was considered to be a relatively attenuated strain of WNV [KUN2011 was considerably more neuroinvasive than the prototype WNVKUN and WNVKUN2009 in both weanling and young adult mice  of the virus, population density, and host feeding behaviour [Cx. annulirostris to be 70-85%, so it has been estimated that < 20% of the population survive the 8–10 day EIP to transmit MVEV, another important encephalitic flavivirus in Australia [KUN2011 being expectorated, it is likely that only a very small proportion of the Cx. annulirostris population would survive to transmit at these time points in the field, potentially offsetting the field impact of our laboratory observation.In addition to increased transmissibility of WNVehaviour . Becauseustralia -33. ConsCx. annulirostris. Thus, it was not surprising that there was an explosive increase in density of this species, with Cx. annulirostris dominant in trap collections that yielded over 10,000 mosquitoes per trap [Cx. annulirostris blood meals from the Rufous Night Heron, Nycticorax caledonicus [Cx. annulirostris is an opportunistic blood feeder [The significantly higher than average rainfall experienced during 2010 and 2011 produced widespread freshwater larval habitats suitable for per trap . Widesprper trap . Analysiedonicus , so the d feeder , this spCx. australicus did not have increased vector competence for WNVKUN2011 compared to WNVKUN2009 and had a significantly lower transmission rate for both virus strains than Cx. annulirostris. Although no isolates of WNVKUN2011 were obtained from Cx. australicus during the 2011 outbreak, this species has yielded WNVKUN isolates previously [Cx. australicus feeds predominately on birds [In our experiments, eviously . Despiteon birds ,37 it maon birds ,39.KUN2011 in Cx. annulirostris, it is possible that other mosquito species may have contributed to the 2011 epidemic. Another member of the Cx. pipiens group, Culex quinquefasciatus is a highly efficient vector of WNVNY99 [KUN2011 outbreak [Culex molestus, this species, as part of the Cx.pipiens group globally, is widely distributed within the region of WNVKUN activity [Aedes, such as Aedes theobaldi, Aedes vittiger, Aedes eidsvoldensis and Aedes sagax were also abundant during the 2010–2011 season [KUN2011 and related flaviviruses.Although the primary objective of the current study was to characterize WNV WNVNY99 , exhibit WNVNY99  and was outbreak . While tactivity  and baseactivity . Finally1 season  and may KUN2011 was unprecedented. It was geographically widespread and WNVKUN activity appeared for the first time near populous centers of the east coast. Above average rainfall again triggered high mosquito population densities in some inland locations during the 2011–2012 season [KUN isolate showed similar levels of virulence asWNVKUN2011 in mice , suggesting continued circulation of this pathogenic strain.The 2011 outbreak of equine encephalitis attributed to infection with WNV2 season . However2 season . A 2012 KUN2011 in Cx. annulirostris, we examined susceptibility of this species to infection, replication dynamics and ability to transmit the virus. One of the key findings of the study was more efficient transmission of this virus strain at the later days post infection, when compared to the less pathogenic WNVKUNV2009 strain. Because WNV transmission cycles involve a complex interaction between the virus, vertebrate host and mosquito vector [KUN2011 by Cx. annulirostris, elevated mosquito populations, an abundance of ardeid birds serving as amplifying hosts, opportunistic blood feeding habits of the key vector and increased pathogenicity of the virus in horses may have driven the 2011 equine epidemic in southeastern Australia.To assess the relative fitness of WNV


From food to fertilizer, algal derived products are largely employed in assorted industries, including agricultural, biomedical, food, and pharmaceutical industries. Among different chemical compositions isolated from algae, polysaccharides are the most well-established compounds, which were subjected to a variety of studies due to extensive bioactivities. Over the past few decades, the promising results for antiviral potential of algae-derived polysaccharides have advocated them as inordinate candidates for pharmaceutical research. Numerous studies have isolated various algal polysaccharides possessing antiviral activities, including carrageenan, alginate, fucan, laminaran, and naviculan. In addition, different mechanisms of action have been reported for these polysaccharides, such as inhibiting the binding or internalization of virus into the host cells or suppressing DNA replication and protein synthesis. This review strives for compiling previous antiviral studies of algae-derived polysaccharides and their mechanism of action towards their development as natural antiviral agents for future investigations. Distinctive structure of viruses and their complicated life cycle have made the discovery of definite treatments against antiviral infections extremely demanding. Despite comprehensive studies for suitable vaccines and treatments against viral infections over the past half of a century, still several infections, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and dengue virus (DENV), afflict a substantial proportion of the world populations in all generations –3. VacciThe unique living environment has gifted the marine world an assorted collection of algae from microorganisms to giant seaweeds. Various types of algae from microscopic diatoms to unicellular organisms and seaweeds reaching 30 m in length have stimulated significant economic interest as agar, fertilizer, food, source of iodine, and potash . AmongstPolysaccharides, also known as glycans, are the most abundant form of carbohydrate materials in the nature. Due to marked biological activities, numerous studies have been performed on different algae-derived polysaccharides, namely, agar, alginate, fucoidan, carrageenan, laminaran, proteoglycans, galactosyl glycerol, and rhamnan sulfate , 17–20. A study by Gerber and colleagues in 1958 which showed inhibition of mumps and influenza B virus by polysaccharides from marine algae has introduced algae-derived polysaccharides as a potent source of antiviral agents . Subsequ Chondrus, Gigartina, Hypnea, and Eucheuma, wherein they serve a structural function similarity to that of cellulose in plants , an, an Chon in vitro by inhibiting the initial stage of infection , 55 Lami in vitro and in vivo. These special effects revealed that 911 drug inhibited the viral replication of HIV via significantly decrementing the activity of reverse transcriptase (RTase), discontinuing the virus adsorption, and improving the defense mechanisms of the host cells .–72.70–72 in vivo and in vitro, including important human pathogens such as HIV, HSV1-2, dengue virus, and cytomegalovirus -β-d-Glcp-(1 → 4)-d-Galp-(1→). Kanekiyo and colleagues identified that nostoflan has a noble inhibitory effect on several enveloped viruses including HSV-1 and HSV-2, human cytomegalovirus, and influenza A virus. The most sensitive stage of viral replication to nostoflan in time of addition during experiment was assumed to be the initial stage of infection covering the virus binding or/and internalization processes. But further investigation confirmed that uniquely the inhibition of virus binding to host cells was responsible for the antiherpetic effect induced by nostoflan and their findings recommended nostoflan as a great antiherpes candidate [Nostoflan (NSF) is an acidic polysaccharide found in edible blue-green alga,andidate . Schizymenia pacifica. SAE is a member of λ-carrageenan, which is composed of galactose (73%), sulfonate (20%), and 3,6-anhydrogalactose (0.65%), and it is known that SEA is a selective inhibitor of HIV reverse transcriptase and replication in vitro and has no adverse effect on cell proliferation [ in vitro. Accordingly, it is unlikely to have an adverse effect on the proliferation of cell culture. It is hypothesized that sulfate residues play a vital role in RT suppression activity. This hypothesis is substantiated by inducing activity in polysaccharides [Sea algae extract (SAE) is a sulfated polysaccharide with the high molecular weight isolated from marine red algaferation . Similarcharides . in vitro investigation in a single host cells. However, in vivo examination is pivotal for the development of pharmaceutical drugs. In addition, further studies including clinical trials to exploit the antiviral activity against viral infections in human are another area for further research. To the best of our knowledge, the synergistic activity of algal polysaccharides against viral infections was not adequately covered in previous studies. It is hoped that this review would be a source of enlightenment and motivation for the interested researchers to conduct further in vitro, in vivo, and clinical analyses on the antiviral activity of algal polysaccharides with a view to developing new antiviral drugs.A variety of biological activities are reported for algal polysaccharides including the transcendent antiviral effect. In the majority of cases, the antiviral activity of these polysaccharides is exerted through suppression of virus adhesion to the host cells. Throughout our literature review, it was noticed that despite numerous investigations which substantiated the antiviral potential of algal polysaccharides, few aspects may have been neglected by researchers to date. Many of the reported pharmacological studies were only limited to


Schistosoma haematobium eggs can manifest as different lesion types visible by colposcopy; rubbery papules (RP), homogenous sandy patches (HSP) and grainy sandy patches (GSP). Pronounced tissue eosinophilia is a candidate marker for active S. haematobium pathology, as viable schistosome egg granulomas often are eosinophil rich. Here it was investigated whether eosinophil granule proteins ECP (eosinophil cationic protein) and EPX (eosinophil protein-X) in urine and genital lavage can be used as markers for active FGS lesions.Genital granulomas induced by Uro-genital samples from 118 Malagasy women were analysed for ECP and EPX by standard sandwich avidin/biotin amplified ELISA.The women with RP lesions had significantly higher levels of ECP and EPX in both lavage and urine. Furthermore, women with RP lesions were significantly younger than those with GSP. This could indicate that RP lesions might be more recently established and thus represent an earlier inflammatory lesion stage.ECP in genital lavage might be a future tool aiding the identification of FGS pathology at a stage where reversibility remains a possibility following praziquantel treatment. Schistosoma haematobium produce eggs which can inflict lesions both in the urinary and genital tract. Lesions in the female genital tract have been hypothesised to confer higher risk of contraction of HIV and other genital infections. These epithelial genital lesions are visibly different and three types can be observed; rubbery papules, homologous sandy patches and grainy sandy patches. Like other helminths, active S. haematobium infection is associated with eosinophilia. Therefore eosinophil granule proteins might be useful markers for active inflammation related to female genital schistosomiasis lesions. This study identifies the rubbery papules as a potential early-stage inflammatory lesion type associated with high levels of eosinophil granule proteins in vaginal lavage. It may be advantageous to identify female genital lesions relatively early after infection as chronic inflammation stage lesions might not respond to praziquantel treatment.The blood-dwelling fluke  Schistosoma haematobium affects millions of people in the Sub-Saharan region resulting in a substantial morbidity burden ranging from subtle to severe in individuals Schistosoma haematobium eggs not only in the urinary tract but also the genital tract tissues is far from a new finding, as it was first described in Egypt in 1899 S. haematobium endemic areas The poverty associated disease uro-genital schistosomiasis caused by S. haematobium in the Sub-Saharan region of which a large proportion is at risk of having genital morbidity Approximately 112 million people are estimated to be infected with S. haematobium egg induced granulomas are dominated by eosinophils It has been well documented in studies focusing on the urinary tract that the S. haematobium prevalence) in previous surveys conducted in 2009 by IPM in collaboration with the schistosomiasis unit at the Madagascar Ministry of Health. These surveys showed a S. haematobium eggs in urine prevalence of 82% (61/74) in one village and 84% (89/106) based on pooled data from two high endemic villages. In the last high endemic village the prevalence of S. haematobium was based on observation of a high prevalence of macro-haematuria as reported by the local medical officer. The fifth selected village had low S. haematobium endemicity  based on an IPM survey from 1999 which found 8% (8/96) S. haematobium egg positive in urine. All five villages had comparable socio-economic levels and infrastructures. Inclusion criteria for study enrolment were 15–35 years of age, female gender, not having received anti-schistosomal treatment within the previous two years and having lived in the district for more than five years. Exclusion criteria were pregnancy, virginity, and eggs in urine status. One hundred and eighteen women, who wanted to participate and met the inclusion criteria, were recruited. Samples were obtained from 79 women from the four hyper-endemic villages . The control group consisted of samples from 39 women from the low endemic village matched by age to the women from the S. haematobium high endemic villages. Only control group individuals who did not shed eggs in the collected urine samples were recruited.A FGS case definition study was carried out in June–July 2010 in Miandrivazo district, Madagascar headed by the Institut Pasteur de Madagascar (IPM). The study participants were recruited from five villages in Miandrivazo district, which is located in the western part of Madagascar. Four of the chosen villages were identified as potential hyper-endemic villages (>50% S. haematobium arithmetic mean egg counts (eggs/10 ml urine) were calculated based on the three urine samples. The urine sample for ECP and EPX level was collected on the day of the pelvic examination. An aliquot of 2 ml urine was immediately snap-frozen in liquid nitrogen. Urine samples were kept at −80°C and transported to the laboratory on dry ice. Furthermore, genital lavage was collected by spraying 10 ml 0.9% NaCl on the cervix and the inner half of the vaginal surfaces, whereafter it was pulled back into the syringe and re-sprayed. This was repeated four times in total. A set of 2 ml vials for ECP and EPX level determination were frozen and kept at −80°C from collection to the laboratory.Midstream urine samples were collected between 9am and 2pm on three consecutive days one of which coincided with the pelvic exam. A subsample of 10 ml urine was filtrated through a Nucleopore membrane immediately after collection and subsequently counted by standard light microscopy in the field laboratory. As briefly outlined, FGS was diagnosed by colposcopy as previously described by Kjetland et.al. Trichomonas vaginalis , Neisseria gonorrhoea (PCR), Chlamydia trachomatis (PCR), Mycoplasma genitalium (PCR), Human papilloma virus (High-risk-HPV PCR), Candida albicans  and bacterial vaginosis (microscopy of wet-mount (Amsel's criteria) and Pap-stained slides). If a genital ulcer was found a swab was applied on the ulcer to test for Herpes simplex virus type 2 (PCR), and Haemophilus ducreyi (PCR). Furthermore cytobrush and spatula were used to collect material for Papanicolaou-stained slides in order to check for S. haematobium eggs and inflammatory signs. Lastly a serum sample was also tested for Treponema pallidum .Vaginal swabs were taken in order to test for a range of genital infections. The diagnostic methods used to supplement the clinical findings for diagnosis are given in brackets; et. al. Standard curve detection range was 31–2000 pg/ml for EPX and 16–1000 pg/ml for ECP. Plates were read at 492 nm with a reference reading at 595 nm in ELISA-reader (Thermo Scientific Multiskan FC).Urine and genital lavage samples were analysed for ECP and EPX level in 2012 by standard avidin/biotin amplified sandwich ELISA using the protocol developed by Claus Reimert Ethical permission was granted by the Committee of Ethics at the Ministry of Health in Madagascar (N° 031-CE/MINSAN 4 June 2010). A female physician informed the study participants in the local Malagasy language about the purpose and overview of the study, the procedures, the benefits and possible negative consequences of participation, privacy and confidentiality procedures, and the right to ask questions and to withdraw at any time point. The woman was asked for a signature if she accepted to participate in the study. Illiterate women who gave their free consent were asked to stamp their fingerprint. This was a procedure approved by the Committee of Ethics.All women diagnosed with schistosomiasis were treated with a single dose of praziquantel (40 mg/kg body weight). If symptoms or signs of sexually transmitted infections or other diseases were observed, treatment or referral in accordance with the standard syndromic approach used in Madagascar was applied free of charge. Asymptomatic STIs were treated upon reception of STI testing results from the laboratory. Partner treatment was offered by a male doctor in a neighbouring location. All study participant information was anonymised and securely stored.ELISA absorbance readings and protein concentration calculations were handled using SkanIt3.1.0.4 software. Only standard curves with a correlation co-efficient (r) of minimum 0.95 were accepted, and protein concentrations were determined using linear regression. Data were analysed using SPSS 19.0.0 for Windows (IBM SPSS Inc.), and the graphical representation in UniProtKB/Swiss-ProtECP: P12724EPX: P10153S. haematobium egg counts in the range from 1–2463 eggs/10 ml urine with an geometric mean intensity of 35 eggs/10 ml urine and a median of 51 eggs/10 ml urine. Gynaecological examinations showed that 41 of the total 118 women were FGS positive (34.7%), whereas 77 had neither a rubbery papule (RP), homogenous sandy patch (HSP), nor a grainy sandy patch (GSP) on the vaginal or cervical mucosal surface visible by colposcopy as described in Kjetland et.al. 2005. Women from the low endemic village (n = 39) were recruited based on being S. haematobium egg negative in urine, however, 10 women (25.6%) from this group were found FGS positive (Women from the hyper-endemic villages (n = 79) had positive . No urinThe age span of the 118 women was from 15 to 35 years of age and there was no significant difference in age between FGS positives and negatives (p = 0.719). However, women with only GSP lesions were significantly older than RP positive women (p = 0.007).12 ng/ml), which was also higher than the level in FGS negative women of 6.4 ng/ml ECP (range 0.01–1720 ng/ml). However, this difference was not significant (p = 0.056). Positive correlations were found between the mean S. haematobium egg count in urine and the levels of ECP in both genital lavage and urine (p<0.01). However, the correlation was weaker in genital lavage (r = 0.257) as compared to urine (r = 0.773).FGS positive women had a median level of 72.5 ng/ml (range 2.79–5480 ng/ml) of ECP in genital lavage. This significantly (p = 0.002) exceeded the ECP level found in genital lavage in the FGS negative women of 24.4 ng/ml (range 0.06–625 ng/ml). In urine FGS positive women had a median ECP level of 56.1 ng/ml (range 0.03–1.27S. haematobium egg count in urine (p<0.01), but the strongest correlation was found in urine (r = 0.550) compared to genital lavage (r = 0.403).The median level of EPX in genital lavage was 63.5 ng/ml (range 0.92–3950 ng/ml) in FGS positive women, which was significantly (p = 0.003) higher, than the median level found in the FGS negative group of 10.6 ng/ml for EPX (range 0.12–810 ng/ml). In urine from FGS positive women the median level of EPX was 1650 ng/ml (range 103–25400 ng/ml). FGS negative women had a median level of 875 ng/ml (range 40–8840 ng/ml) in the urine, which was significantly lower compared to the FGS positive women (p = 0.024). EPX levels in both genital lavage and urine were positively correlated to the mean In order to investigate whether the significantly elevated ECP and EPX levels found in women with FGS were associated with a particular type of lesion pathology, analysis was done stratified by lesion categories . A signiTrichomonas vaginalis, Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoea, bacterial vaginosis, Candida albicans or high-risk Human papilloma virus) and age. Test for Haemophilus ducreyi and Herpes simplex virus type 2 were only carried out if a genital ulcer was suspected , hence these variables could not be taken into account.Multinominal logistic regression show that having RP pathology encompasses a three-fold risk of elevated ECP  and EPX  levels in genital lavage. The regression analysis was controlled for presence of other genital infections  type lesion. In contrast sandy patches (grainy or homogeneous) were not associated with significantly higher granule protein levels in genital lavage. This could indicate that RPs represent eosinophil rich lesions, whereas GSPs and maybe HSPs might represent a later stage of the disease or at least a different lesion type in terms of inflammation. This corresponds with previous findings showing that high ECP levels in lavage were associated with FGS The likelihood of contracting schistosomiasis depends on water contact. Infection intensity measured by urinary egg output normally peaks in 10–14 year olds where after it declines in adults depending on the level of transmission S. haematobium eggs, which excrete antigen. Presence of antigen may spark local immune reactions resulting in activated endothelial cells and chemotactic recruitment of a range of immune cells. In vitro studies have demonstrated that soluble egg antigens (SEA) from S. mansoni eggs induce endothelial cell proliferation and activation and it is possible that viable S. haematobium eggs in a similar manner induce vascular activity S. haematobium granulomas containing viable eggs is the recruitment of high eosinophil numbers, but also enhanced macrophage, fibroblast and endothelial cell activity has been demonstrated S. haematobium eggs If RPs are in fact early-stage lesions, it is very likely that they contain viable S. haematobium egg induced RP pathology, since basophils and particularly neutrophils occasionally might present a bias as source of the granule proteins Some caution is necessary when interpreting ECP/EPX levels as measures for tissue eosinophilia related to S. haematobium infection Both ECP and EPX protein levels were higher in association with RPs in genital lavage, but ECP in appears to be the better candidate marker, since EPX levels were higher in urine than in genital lavage even in the control group. Likewise, previous reports on urine samples from Kenyan children point towards ECP being superior to EPX as eosinophil marker protein for Studies conducted in Egypt, Ghana, Madagascar, Malawi, South Africa and Zimbabwe point towards a significant and under-recognised FGS related morbidity which may manifest as diversely as pelvic pain, infertility, a 3-fold increased risk of HIV as well as various social consequences


We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula. Camelus dromedarius) were implicated for the first time as a possible source for human infection on the basis of the presence of MERS-CoV neutralizing antibodies in dromedaries from Oman and the Canary Islands of Spain , was identified as the cause of severe respiratory disease in humans during 2012 , severe acute respiratory syndrome CoV (residues 1–676), and human CoV OC43 (residues 1–760) by using extensively validated protein-microarray technology, as described . All proSince the discovery of MERS-CoV in 2012, accumulating serologic and molecular evidence demonstrates that the virus in dromedaries is genetically very similar to MERS-CoV in humans and points to the conclusion that dromedary camels are reservoirs for human infection. MERS-CoV genomic fragments have been detected in dromedaries in Qatar (Samples in this study were collected during 2009–2011, confirming observations by us and others ; Table 1A question raised by these findings is whether human cases occur outside the Arabian Peninsula and if such cases are currently underdiagnosed in Africa. In addition, for the whole region, the possibility exists that MERS-CoV illness occurred before its discovery in 2012 and that such infection has been overlooked in the areas with evidence for virus circulation among animals during the past 10 years. Retrospective studies of cohorts of humans with respiratory illnesses of unknown etiology should address this notion. Alternative explanations for the lack of cases in Africa could be the following: a different risk profile, for instance, related to demographics and local practices; or subtle genetic differences in the circulating virus strain. Full-genome sequencing, virus isolation, and phenotypic characterization of viruses circulating outside the Arabian Peninsula will resolve this issue. Meanwhile, awareness of MERS-CoV infections should be raised among clinicians in Africa.


Chlamydia muridarum is known to colonize in the gastrointestinal tract for long periods of time, which has been hypothesized to serve as a reservoir for spreading to the genital tract. To test this hypothesis, a luciferase-expressing C. muridarum was used to establish a long-lasting infection in the mouse gastrointestinal tract following either intragastric or intrarectal inoculations. In vivo imaging revealed significant bioluminescent signals mainly in the mouse abdominal area throughout the experiments. Ex vivo imaging localized the signals to the mouse gastrointestinal tract, which was confirmed by monitoring the C. muridarum organisms in the mouse organs/tissues. Despite the long-lasting colonization in the gastrointestinal tract and active shedding of infectious organisms in the rectal swabs, the organisms did not cause any significant infection or pathology in the genital tract throughout the experiments, which was reproduced in multiple strains of mice and with an increased inoculation dose to the gastrointestinal tract. The above observations have demonstrated that the long-lasting C. muridarum organisms from the gastrointestinal tract are inefficient in auto-inoculating the genital tract, suggesting that the gastrointestinal tract Chlamydia may utilize an indirect mechanism to affect its pathogenicity in the genital tract. Chlamydia has been detected in the gastrointestinal (GI) tracts of animals . The homogenates were further briefly sonicated and spun at 3000 rpm for 5min to pellet remaining large debris. The supernatants were titrated for live C. muridarum organisms on HeLa cells as described above. The results were expressed as log10 IFUs per organ or tissue segment.For quantitating live organisms from mouse organs and tissue segments, immediately after 10 IFUs per sample.The immunofluorescence assay used for titrating live organisms were carried out as described previously , 28. ForC. muridarum, a portion of each tissue homogenate was transferred to the lysis buffer provided with Quick-gDNA miniPrep Kit  and subjected to DNA extraction following manufacturer’s instruction. Each DNA preparation was eluted in 100μl elution buffer, and 2μl was used for quantitative PCR (qPCR). The following primers derived from the Chlamydia 16S rRNA coding region were used: forward primer (5′-CGCCTGAGGAGTACACTCGC-3), reverse primer, (5′-CCAACACCTCACGGCACGAG-3‘) and Double-Quenched Probe (5′-CACAAGCAGTGGAGCATGTGGTTTAA-3′) . PCR was carried out in a total volume of 10 μ in a CFX96 Touch Deep Well Real-Time PCR Detection System with iQ Supermix real-time PCR reagent . Genome copy numbers for a given sample in triplicate were calculated based on a standard plasmid DNA prep in the corresponding samples. The qPCR conditions included an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute.To quantitate the genome copies of Seventy days after infection, mice were euthanized for harvesting genital tracts. Gross pathology of hydrosalpinx was observed and documented by high-resolution digital photography. Hydrosalpinx was scored according to an ordinal scale where 0 indicates no hydrosalpinx, 1 indicates hydrosalpinx that is only observable under magnification, 2 indicates visible hydrosalpinx smaller than the size of the oviduct, 3 indicates hydrosalpinx roughly equal to the size of the oviduct, and 4 indicates hydrosalpinx larger than the oviduct. Bilateral hydrosalpinx severity was calculated for each mouse as the summed scores of the left and right oviducts. Hydrosalpinx incidence was calculated as the number of mice with a bilateral score of 1 or higher divided by the total number of mice in the group. Chronic inflammation or infiltration of mononuclear cells into the genital tract tissues was evaluated histologically. Following gross pathology assessment, whole genital tracts were fixed in 10% neutral formalin, stored in 70% ethanol in water, embedded in paraffin, and serially sectioned longitudinally across the plane of the whole genital tract at 5-μm intervals on an AccuCut SRM 200 microtome . Three nearly equidistant sections at roughly 5 μm, 30 μm, and 55 μm into the lateral surface of the oviduct were subjected to hematoxylin and eosin (H&E) staining, sealed on glass tissue slides, and observed under a microscope for infiltration of mononuclear inflammatory cells into tissue surrounding the oviduct. These infiltrates were scored on an ordinal scale where 0 represents no cellular foci, 1 indicates a single focus, 2 indicates two to four loci, 3 indicates more than four foci, and 4 indicates confluent infiltration. The median of the three scores served as a single value for each oviduct unilateral inflammation score, and both unilateral scores for each mouse were combined to form a bilateral score. Bilateral oviduct dilation scores were acquired from the same three sections of each oviduct in the same manner as the inflammation scores but using the criteria for scoring hydrosalpinx described above.The incidence rates were analyzed using Fisher’s exact test while the number of live organisms (IFUs), genome copies and pathology scores were analyzed using the Wilcoxon rank sum test for comparing any two groups and Kruskal-Wallis for comparing more than two groups.in vivo imaging technology for monitoring the distribution of the luciferase-expressing C. muridarum organisms in CBA/J mice following an intragastric or intrarectal inoculation (C. muridarum organisms were restricted to the gastrointestinal tract only or also spread to other organs/tissues, we monitor the organism distribution by both using an ex vivo imaging of the mouse organs and directly detecting the C. muridarum organisms in the corresponding organs/tissues on days 28 and 70 after the initial inoculation (ex vivo imaging, we found that the bioluminescence signals were only from the GI tract tissues, such as the rectum, cecum and stomach. Homogenates made from the same organs/tissues, together with the rectal and vaginal swabs, were titrated for both C. muridarum genomes using qPCR and live organisms using an immunofluorescence assay. We found that significant levels of C. muridarum genomes and live organisms were only detected from the GI tissues/swabs. Together, the above observations have demonstrated that the long-lasting persistence of C. muridarum organisms is restricted to the GI tract only.We used a whole body culation . The lucculation . When alC. muridarum infection in the GI tract can serve as a reservoir for auto-inoculating the genital tract, we carefully monitored both the GI tract and genital tract chlamydial organism shedding over time from the CBA/J mice inoculated with C. muridarum via intragastric, intrarectal or intravaginal routes (C. muridarum organisms did not effectively auto-inoculate the genital tracts. The minimal levels of live organisms detected in one or two vaginal swabs harvested on day 7 or 14 may be caused by swabbing contamination since the chlamydial organisms were not detected in any two consecutive swabs from the same mice. On the contrary, the intravaginally inoculated mice displayed robust live organism recoveries in both the rectal and vaginal swabs and the live organisms continued to shed in the rectal swabs even after the vaginal swabs were cleared, which is consistent with what we have previously reported [C. muridarum organisms can readily spread from the genital tract to the GI tract [7 IFUs of C. muridarum. We further compared both the macroscopical (C. muridarum into the GI tract developed any hydrosalpinx. At the microscopic level, no significant inflammatory infiltration was observed in the genital tissues of the mice with the GI tract-inoculation although significant inflammatory infiltration was detected in vagina, uterine and oviduct tissues of the intravaginally infected mice. To test whether the lack of autoinoculation detected in the CBA/J mice is only restricted to this strain of mice or a universal phenomenon, we repeated the experiments in both C57BL/6J and C3H/HeJ mice (C. muridarum organisms via an intrarectal route and monitored for the presence of live organisms in the rectal and vaginal swabs. Significant levels of live C. muridarum organisms were only detected in the rectal but not vaginal swabs. Again, the scattered sheddings detected in the vaginal swabs might be caused by swabbing contamination. More importantly, no hydrosalpinx was detected in any of these mice. Thus, the lack of autoinoculation from the GI tract to the genital tract was reproduced in multiple strains of mice.To address whether the long-lasting l routes . The intreported . It is cGI tract , these oscopical  and micrscopical  pathologHeJ mice . Both stC. muridarum organisms can last for long periods of time in the mouse GI tract [C. muridarum can persist for long periods of time only in the GI tract and whether the long-lasting C. muridarum organisms in the GI tract can effectively auto-inoculate the genital tract and contribute to the C. muridarum pathogenicity in the upper genital tract. We have shown that after the luciferase-expressing C. muridarum organisms were introduced into the GI tract via either intragastric or intrarectal inoculation, the organisms only colonized the GI tract for long periods of time. First, the whole body in vivo imaging revealed that the luciferase-generated signals were restricted to the abdominal area throughout the experiments; Second, the ex vivo imaging of all organs only detected the bioluminescent signals in the GI tract on days 28 and 70 after the inoculation; Finally, the C. muridarum organisms were detected only in the GI tract but not other organ tissues on days 28 and 70, confirming the specificity of the luciferase-generated bioluminescent signals. These observations together have both confirmed previous observations that C. muridarum infection in the GI tract can last for long periods of time [C. muridarum via either intragastric or intrarectal routes can only lead to a long-lasting infection in the GI tract but not other organs.The GI tract –9. In th of time –9 and deC. muridarum in the mouse GI tract via either intragastric or intrarectal inoculation and confirmed that the long-lasting C. muridarum in the GI tract is only restricted to the GI tract. We then monitored the genital tract infection and inflammation in mice that continuously shed live infectious organisms from their rectal swabs. We found no significant levels of C. muridarum organisms from the vaginal swabs of the same mice. More importantly, no significant pathology and inflammation were detected in the genital tract of these mice. These observations were reproduced in different strains of mice infected with the C. muridarum G13.32.1 clone. It is clear that active shedding of live C. muridarum organisms from the GI tract not necessarily auto-inoculate the genital tract in mice. Although minimal levels of live organisms were detected in the some vaginal swabs, these incidental positive detections might be caused by the swabbing contamination because the positive detection was rarely found in two or more consecutive swabs from the same mouse. More importantly, these mice did not show any sign of inflammation in the genital tract tissues.Chlamydia has been detected in the GI tracts of animals  and huma7 IFUs of C. muridarum were delivered to mouse GI tracts, the levels of live organism shedding remained the same as those from mice intrarectally inoculated with 5 x 104 IFUs, suggesting that an inoculation dose of 5 x 104 IFUs was already at the saturation level and was sufficient for achieving a maximal level of infection in the mouse GI tract. It is not clear why the mouse GI tract was saturated by an inoculation dose of 5 x 104 IFUs. The saturation can be caused by either the limited number of cells available for C. muridarum infection or an active suppression of C. muridarum replication in the mouse GI tract. Nevertheless, mice inoculated with either 5 x 104 or 1 x 107 IFUs developed similar but significant levels of live organism shedding in the rectal swabs throughout the experiments. These continuous live organism sheddings from the GI tracts did not cause any significant infection in the genital tracts, suggesting that under the experimental condition, the GI tract chlamydial organisms even at their maximal shedding levels were not able to autoinoculate the genital tracts.Besides reproducing the lack of autoinoculation observation in multiple strains of mice, we also tested whether an increased inoculation dose to the mouse GI tract could facilitate autoinoculation. However, when 1 x 10C. muridarum into the genital tract was only based on the C. muridarum murine model. We should be cautious in applying the knowledge learnt from mice to C. trachomatis infection in humans. This is because not only the biology of both the organisms and hosts is different, but also the human behaviors differ significantly from those of mice. For example, if a person use fingers to touch both the anorectum and vagina, the chance of chlamydial spreading from the GI to genital tract is high. To evaluate whether this route of chlamydial spreading can enhance chlamydial pathogenicity in the genital tract, sexual behaviors-related clinical studies should be carried out.The conclusion on the lack of autoinoculation by the GI tract C. muridarum from the GI into the genital tracts as demonstrated above does not necessarily mean that the GI tract Chlamydia cannot contribute to the chlamydial pathogenicity in the upper genital tract. On the contrary, what we have learnt from the current study may guide us to the correct direction for further investigating chlamydial pathogenesis. We hypothesize that besides the direct autoinoculation, indirect mechanisms may play significant roles. For example, the long-lasting pathology in the upper genital tract is accompanied with chlamydia-specific host responses [It is worth noting that the lack of effective autoinoculation of esponses , 37, 38.


Zika virus currently poses a global threat and is a major public health issue throughout Latin America and the Caribbean. However, Zika virus infections in humans have also been observed in other regions, including Southeast Asia, where arboviral diseases are very common. In this study, we summarize the current status of Zika virus in Southeast Asia. This review aims to provide an overview of the current situation and also to suggest ways of adequately managing the emergence of Zika virus in Southeast Asia.The literature searching for the reports on Zika virus in Southeast Asia was done using standard database PubMed and the re-analysis and summarization on the reports was done.A limited number of reports have addressed Zika virus disease in Southeast Asia, but it is has been confirmed that a problem already exists. Individual case reports and outbreaks of Zika virus have been confirmed in Southeast Asia. Several reports have also described patients becoming infected after visiting Southeast Asia. In addition, the concurrent circulation of Zika virus with other arboviruses has been confirmed.As a tropical region with a high prevalence of arboviral diseases, the emergence of Zika virus in Southeast Asia is a major concern. It is essential for local medical personnel to recognize this disease. Given the status of Southeast Asia as a globally important tourist destination, continuous updates on the status of Zika virus in Southeast Asia are required and should be incorporated into global health advisories regarding travel. Zika virus was discovered in 1947 in Uganda, and has subsequently emerged as a new disease . InitialHowever, greater levels of concern regarding this infection developed due to a recent outbreak on the Yap Islands in Micronesia. The Zika virus then spread to French Polynesia and other Pacific regions, causing several outbreaks in 2013 and 2014 . This fiAt present, Zika virus disease is a major public health issue throughout Latin America and the Caribbean. However, Zika virus infections have also been observed in other regions, such as Southeast Asia, where arboviral diseases are very common. However, fewer cases have been confirmed than in Latin America. Due to the clinical similarity of Zika virus infections with both dengue and chikungunya fever, which are prevalent in this area, it is possible that Zika virus infections are underdiagnosed . This reAedes species) that are also able to transmit Zika virus; therefore, it is clear that Zika virus can be transmitted in this region and may emerge as a new major problem. Southeast Asian countries may be the next major foci of Zika virus outbreaks. The data on the present status of the disease in this area are also useful to enhance global monitoring of the disease. In this section, the situation in each Southeast Asian country is summarized.Southeast Asia is a tropical region where many tropical diseases are endemic. In particular, the arboviral diseases dengue and chikungunya fever are highly prevalent in this area. The main reason for this is the abundance of mosquito vectors  in Indonesia. However, animal bites have never been proven to be a mode of transmission of Zika virus. The case reported by Leung et al. [Moreover, reports have described travelers presenting with Zika infections after returning from Indonesia ,14. KwonAedes aegypti and concluded that the mosquitoes are “susceptible to and are potentially capable of transmitting” Zika virus. Wong et al. [Ae. albopictus to transmit the virus and proposed “the possibility that the virus could be established locally.” Based on these data, strict mosquito control must be implemented in Singapore. Although no cases of human infections have been reported, Zika virus infections pose a risk in Singapore. Nevertheless, Li et al. [Singapore is the smallest country in Southeast Asia. Similarly to other Southeast Asian countries, arboviral diseases are common in Singapore. Some interesting reports have recently described the vectors of Zika virus in Singapore ,16. Li ei et al.  claimed Myanmar is a developing country in Southeast Asia, with a high poverty rate and poor public health system. Arboviral diseases are very common in Myanmar, but few data have been published regarding their prevalence . No repoSimilarly to Myanmar, the Lao People’s Democratic Republic  is a poor country in Indochina with limited resources for public health. Arboviral diseases are also common in Laos . SimilarCambodia is a country in Indochina where arboviral diseases are highly prevalent . Many reThailand is a country in Indochina located next to Cambodia. Some case reports of Zika virus infections in Thailand have been published, and it was confirmed that the virus originated from Cambodia ,22. The Zika virus has also been reported in travelers returning from Thailand . The firVietnam is a country in Indochina where arboviral diseases are common . No repoBrunei is a small country in Southeast Asia. No information has been published regarding the presence of Zika virus in Brunei.Timor Leste is a small island country in Southeast Asia. No information has been published regarding the presence of Zika virus in Timor Leste.Malaysia is a tropical country in Southeast Asia where arboviral diseases are very common. The possibility that Zika virus disease could take hold in Malaysia due to an abundance of mosquito vectors was proposed long ago . The virThe Philippines are an island country in Southeast Asia, located close to the Yap Islands in Micronesia, where a major outbreak of Zika virus disease occurred. The Yap Islands are located between the Philippines and Papua New Guinea. Zika virus infections have already been confirmed in the Philippines . A molecThe Andaman/Nicobar Islands are geographically classified as islands in Southeast Asia. No information has been published regarding the presence of Zika virus in the Andaman/Nicobar Islands.Christmas Island is also geographically classified as an island in Southeast Asia. No information has been published regarding the presence of Zika virus on Christmas Island.Although mosquito-borne illnesses are abundant in Southeast Asia, few reports have been published on Zika virus disease in this area. However, Zika virus infections have been confirmed in this region . Zika viAs noted, case reports and outbreaks of Zika virus infections have been confirmed in Southeast Asia. Many reports have also been published describing patients becoming infected after visiting Southeast Asia. Since many tourists visit Southeast Asia annually, awareness of this risk is important and preventive measures must be taken before travel. Given the volume of tourism to Southeast Asia, continuous updates on the status of Zika virus in Southeast Asia are necessary and should be incorporated into global health recommendations for travel.In addition, the concurrent circulation of Zika virus and other arboviruses has been confirmed. As a tropical region with a high prevalence of arboviral diseases, the emergence of Zika virus in Southeast Asia is a major concern. Zika virus infection should be considered both in local cases and in travelers returning from Southeast Asia with clinical features of arboviral disease.