P = 0.001). An upper respiratory tract infection (URTI) significantly increased the risk of COM/ROM . Snoring appeared to be a significant risk factor for COM/ROM . A patient history of acute otitis media (AOM)/ROM increased the risk of COM/ROM . Passive smoke significantly increased the risk of COM/ROM . Low social status appeared to be a risk factor for COM/ROM . Our meta-analysis identified reliable conclusions that allergy/atopy, URTI, snoring, previous history of AOM/ROM, Second-hand smoke and low social status are important risk factors for COM/ROM. Other unidentified risk factors need to be identified in further studies with critical criteria.Risk factors associated with chronic otitis media (COM) and recurrent otitis media (ROM) have been investigated in previous studies. The objective of this study was to integrate the findings and determine the possible risk factors for COM/ROM based on our meta-analysis. A comprehensive search of electronic bibliographic databases  from 1964 to Dec 2012, as well as a manual search of references of articles, was performed. A total of 2971 articles were searched, and 198 full-text articles were assessed for eligibility; 24 studies were eligible for this meta-analysis. Regarding risk factors for COM/ROM, there were two to nine different studies from which the odds ratios (ORs) could be pooled. The presence of allergy or atopy increased the risk of COM/ROM (OR, 1.36; 95% CI, 1.13–1.64;  Chronic otitis media (COM) and recurrent otitis media (ROM) are two of the most common infectious diseases worldwide. COM and ROM affect diverse cultural and racial groups that are distributed in both developing and industrialized countries. A cross-sectional study conducted in nine countries over three continents revealed that disease prevalence is significant enough to be considered for clinical practice Risk factors associated significantly with COM/ROM include ethnicity http://www.wanfangdata.com.cn/) and China National Knowledge infrastructure (CNKI) database (http://dlib.edu.cnki.net/kns50/). Articles from 1964 to Dec 2012 were included in the search. The search was limited to humans and performed with no language restrictions. Reference lists of the relevant original and reviewed articles were evaluated to identify additional studies. We used controlled vocabularies (Explosion mapped searches of MeSH terms or Emtree thesaurus terms) and text words for chronic otitis media, recurrent otitis media, middle ear cholesteatoma, and mastoiditis. Concepts related to “Otitis media” with the subheadings of congenital, epidemiology, genetics, immunology, microbiology and virology for all Mesh terms in PubMed were reviewed. Areas of focus that were chosen for otitis media in Embase were genetics, immunology and hematology, microbiology, otorhinolaryngology, pediatrics and public health. Furthermore, terms indicating risks, such as “risk factors”, “probability”, “odds ratio”, “risk assessment”, “causality”, “epidemiologic factors”, “epidemiology”, “epidemiologic studies”, “multivariate analysis”, “logistic models” and their entry terms were also included  of COM/ROM;The numbers for both controls and COM/ROM cases;Sufficient data are to determine the odds ratio (OR) with 95% confidence intervals (CIs).We excluded descriptive studies, case reports, case series, reviews, letters, commentaries, and studies on the pathogenesis and treatment of COM/ROM. We excluded repeated reports with a small number of participants and these data were included in large studies mentioned above. We excluded the studies of recurrent acute otitis media, congenital cholesteatoma and unclassified OM. Inclusion discrepancy was resolved in joint discussions by the investigators. We appraised the quality of the studies, focusing on the selection of cohorts and assessment of the outcomes.Two investigators, (Yan Zhang and Jin Zhang) independently extracted and registered the data from the eligible publications. The following data from each article was extracted: author, year of publication, risk factor, type of otitis media, study type, age/years of participants, study duration, number of cases, number of controls, total sample size, study location, ethnic group and diagnostic criteria for COM/ROM. All disagreements were resolved through group discussion.2 statistic, which describes heterogeneity across studies. I2 values of <25% and >50% reflects low and high heterogeneity, respectively The meta-analysis was processed using Review Manager 5.1, version: 5.1.6. We estimated the odds ratios (ORs) and 95% confidence intervals (CIs), and the statistical heterogeneity of the studies was assessed before combining the results. Estimates of the risk factors were pooled using a random effects model Of the total 2971 relevant references identified, 198 articles were considered potentially relevant. The excluded references that were considered irrelevant included reviews, letters, commentaries, studies on pathogenesis, pathologies, and treatment, and microbiological studies. A total of 103 case control or cohort studies examined the risk factors of COM/ROM, and 79 studies failed to meet the inclusion criteria for the following reasons: unclear definition of COM/ROM, no classification of OM, no control groups, and inadequate data for abstraction. For repeated studies, we retained the one with the larger sample size. Figure 1 shows the selection flow for this meta-analysis; 24 independent studies met all of the inclusion criteria P = 0.001). A total of four studies investigated the association between upper respiratory tract infection (URTI) and COM/ROM, which includes the presence of cough or rhinorrhea or nasal stuffiness or sore throat or adenoiditis/adenoid hypertrophy. Pooled data from these showed that URTI significantly increased the risk of COM/ROM . A total of two studies showed that snoring appeared to be a significant risk factor for COM/ROM . Pooled data from two studies revealed that a patient history of AOM/ROM increased the risk of COM/ROM ; nine studies investigated parental smoking, exposure to smoking at home and other smokers residing in the same household of frequent visitors. Pooled data showed Second-hand smoke, including the conditions above, increased the risk of COM/ROM . Pooled data from two studies showed low social status as an increased risk factor of COM/ROM .Pooled data from 7 studies indicated the presence of allergy or atopy and increased the risk of COM/ROM , male sex , attending day-care centers , family history of otitis media , low education of the mother , mother's smoking during pregnancy , larger families and more siblings . Pooled data revealed that an association between breast-feeding >6 months and COM/ROM was not statistically significant , neither was an association between breast feeding (yes/no) and COM/ROM . Pooled risk factors for COM/ROM are summarized in The factors that were determined to not be significantly associated with increased risk included chronic nasal obstruction , which includes the presence of cough or rhinorrhea or sore throat, was indicated as a significant prognostic factor for COM/ROM. Studies support that the mucosal condition of ET could be affected by URTI Snoring, defined as the presence of loud snoring at least three times per week, is a common symptom in children and is highly prevalent in children Previous history of AOM/ROM was studied as a predictive factor for COM/ROM. Subjects who experience episodes of AOM/ROM have an increased risk of developing chronic and recurrent middle ear infections.Second-hand smoke has been reported to be associated with increased prevalence of middle ear disease The possibility that COM/ROM is associated with low social status has been debated for a long period of time Sex difference in otitis media risk has been estimated in various studies. Other than a conclusion that the male sex was more likely to suffer from acute otitis media in children Breast-feeding is believed to provide antimicrobial, anti-inflammatory, and immunomodulatory agents that contribute to an optimal immune system Day-care center attendance could increase the risk of children’s exposure to respiratory pathogens. It has been reported to be a significant risk factor for acute respiratory infectious disease in children The causal relationship between other factors, which include chronic nasal obstruction, family history of otitis media, mother's smoking during pregnancy and COM/ROM is not completely established. Association between larger families and more siblings with COM/ROM was not statistically significant.Genetic predisposition is considered to be an important prognostic factor that could influence the risk of otitis media. Previous candidate gene studies associated a number of immune system genes with otitis media, which included TNF-α, IL-6, IL-10, Tlr4, surfactant, CD14, FcγRIIa, IFNγ, Eya4, p73, MyD88, Fas, E2f4, Plg, Fbxo11, and Evi1 Similar to risk factors for genetic predisposition, other risk factors for COM/ROM prevented the data from being pooled in our eligible studies; these include nutritional factors, medication use during pregnancy, ethnicities of Greenland, White, African American, indoor cooking, pharyngeal reflux, and overweight status.The association between gestational age and COM/ROM from the two groups We noticed that the risk factors of sex, attending day-care centers, large families and more siblings have p-values of 0.06, 0.07 and 0.09, respectively. With the application of the 0.05 p-value, which is the conventionally used criterion, no significance was found. Using a cut-off of 10% for significance may ameliorate this problem but could increase the risk of drawing a false positive conclusion (type I error) 2 was applied to test heterogeneity. In our studies of no inconsistency (I2 = 0) or low heterogeneity (I2<25%), using either fixed or random effect models produced identical results and the same direction of effect. The random effect model was the standard approach for the studies of moderate to high I2 values. Some analysts might try to reduce the heterogeneity by limiting the meta-analysis to a smaller more homogeneous study group. However, this could probably result in misleading conclusions if not performed with care or may limit the scope of the meta-analysis and essentially eliminates any useful information In judging the inconsistency of the studies, IA full understanding of the etiologic factors for COM/ROM could be beneficial for the treatment and prevention of the disease. Our study evaluates the risk factors by an objective scientific procedure, meta-analysis, to provide precise causal prophylaxis evidence. Meta-analysis is widely used in medical studies of randomized clinical trials, as well as etiologic factors of the disease. The controversy of meta-analysis is in the homogeneity of the studies. Dickersin and others noted that heterogeneity is not all that bad The risk factors for COM/ROM are closely interrelated. Our meta-analysis identified reliable conclusions that allergy/atopy, upper respiratory tract infection, snoring, previous history of AOM/ROM, Second-hand smoke, low social status are important risk factors for COM/ROM. Other unidentified risk factors investigated in single studies need possible repeated studies with critical criteria to be estimated properly. We suggest that the above COM/ROM risk factors be interfered effectively to prevent and decrease the onset of the disease.Figure S1Funnel plot. Symmetric inverted funnel shape indicates unlikely publication bias.(TIFF)Click here for additional data file.Figure S2Risk factors for COM/ROM. Pooled odds ratios from eligible studies analyzed in the meta-analysis of risk factors for COM/ROM(TIFF)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.Text S1Search strategy.(DOC)Click here for additional data file.


A disease outbreak with dengue-like symptoms was reported in Guangdong Province, China, in October 2010. Testing results confirmed that the pathogen causing the outbreak was chikungunya virus. Phylogenic analysis indicated that this virus was a member of the Indian Ocean clade of the East/Center/South African subgroup of chikungunya virus. Chikungunya virus (CHIKV) is a mosquito-borne virus that causes fever, headache, rash, nausea, vomiting, myalgia, and arthralgia, and has had a major effect on human health .During September 2010, patients reporting an illness with dengue-like symptoms were recorded by local community clinics in the suburbs of Dongguan, Guangdong Province. For epidemiologic investigation, the Guangdong Center for Disease Control and Prevention defined a clinical case of CHIK fever as a case characterized by sudden onset of fever with arthralgia, maculopapular rash, or myalgia. We identified 173 patients  2–93 years of age in 2 adjacent villages who had similar symptoms. More than 85% of the patients were found in these 2 villages in 97 families  was performed to infer possible source of transmission. Specimens were tested by real-time RT-PCR for CHIKV . Seven samples were positive for IgM and 1 sample was positive for IgG . There w2 and observed daily for <7 days for cytopathic effects (CPEs)  . After sPhylogenetic analysis was performed for partial E1 sequences (7 from this study and 24 from GenBank) by using MEGA5 . SequencOn the basis of sequence analysis, the highest degree of identity was observed with outbreak isolates and the E1 sequence from the Thailand strain (FJ882911) isolated in 2009. Paired identity values were 99% at the nucleotide level and 100% at the amino acid level. Nucleotide substitute analysis showed that a common nucleotide substitution was observed at partial E1 gene site 250 (T→C) in outbreak isolates and FJ882911. This substitution was not observed in other analyzed sequences from GenBank. These results suggested that the virus causing this outbreak was likely transmitted from a source in Southeast Asia and probably evolved from a strain that originated in Thailand.CHIKV was not endemic to China before 2010. However, in recent years, CHIKV strains from Southeast Asia with the ECSA genotype have been transmitted by infected persons to Guangdong Province. We report an outbreak of CHIKV fever in China. The low severity of the disease and misdiagnosis of dengue fever has likely encouraged widespread transmission of the virus. High-density mosquito populations and an immunologically uninfected population were 2 contributing factors in this outbreak.


Arthropod-borne viruses (arboviruses) are maintained in nature by cycling between vertebrate hosts and haematophagous invertebrate vectors. These viruses are responsible for causing a significant public health burden throughout the world, with over 100 species having the capacity to cause human disease. Arbovirus outbreaks in previously naïve environments demonstrate the potential of these pathogens for expansion and emergence, possibly exacerbated more recently by changing climates. These recent outbreaks, together with the continued devastation caused by endemic viruses, such as Dengue virus which persists in many areas, demonstrate the need to better understand the selective pressures that shape arbovirus evolution. Specifically, a comprehensive understanding of host-virus interactions and how they shape both host-specific and virus-specific evolutionary pressures is needed to fully evaluate the factors that govern the potential for host shifts and geographic expansions. One approach to advance our understanding of the factors influencing arbovirus evolution in nature is the use of experimental studies in the laboratory. Here, we review the contributions that laboratory passage and experimental infection studies have made to the field of arbovirus adaptation and evolution, and how these studies contribute to the overall field of arbovirus evolution. In particular, this review focuses on the areas of evolutionary constraints and mutant swarm dynamics; how experimental results compare to theoretical predictions; the importance of arbovirus ecology in shaping viral swarms; and how current knowledge should guide future questions relevant to understanding arbovirus evolution. Arboviruses are predominately RNA viruses in the families Flaviviridae, Togaviridae, Bunyaviridae, Rhabdoviridae, and Reoviridae; yet a single genus in the family Orthomyxoviridae (Thogotovirus) and a single DNA virus in the family Asfarviridae (African swine fever virus) are also included among the arboviruses. The fact that these viruses are almost exclusively RNA viruses may be explained by a requirement for significant plasticity in order to succeed in dynamic host environments [−3 to 10−5 errors / nucleotide / round of replication [Arthropod-borne viruses (arboviruses) are unique in that they require cycling between disparate hosts, ronments . RNA-deplication ,3. This,Dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and the Japanese encephalitis serogroup viruses such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV); alphaviruses including Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), Ross River virus (RRV), and Chikungunya virus (CHIKV); and bunyaviruses including Lacrosse virus (LACV), Rift Valley fever virus (RVFV), and California encephalitis virus (CEV). Some human arboviral pathogens such as Colorado tick fever virus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHV), Louping Ill virus (LIV), and Tick-borne encephalitis virus (TBEV), are primarily, if not exclusively, transmitted by ticks. Additional invertebrate vectors including biting midges and sandflies, among others, also have been implicated in transmission of arboviruses with public health significance [blue tongue virus (BTV), African horse sickness virus (AHSV), Vesicular stomatitis virus (VSV), epizootic hemorrhagic disease virus (EHDV), and LIV generally do not cause significant human disease but do cause considerable disease in both wild and livestock populations and consequently have led to significant ecological and economic disruptions [Arboviruses are responsible for causing a significant public health burden throughout the world, with over 100 species of virus having the capacity to cause human disease. Among these, the majority are mosquito-borne viruses including flaviviruses such as ificance . Greaterificance . Most huruptions ,7.O’nyong-nyong virus (ONNV) emerged in Uganda following a 35 year absence and caused widespread disease [Recent arbovirus outbreaks have demonstrated the potential of these viruses to emerge and expand their range, many as a consequence of changing climates and landscapes . The imp disease ; and in  disease . A close disease . Other a disease ,25.These recent outbreaks, together with the continued devastation caused by viruses such as DENV, YFV, and JEV, which remain endemic throughout their geographic range, demonstrate the need to better understand the selective pressures that shape arbovirus evolution and emergence. Specifically, a comprehensive understanding of host-virus interactions and the role of host-specific and virus-specific evolutionary pressures is needed to fully evaluate the factors that govern the potential for host shifts and geographic expansions. One approach to advance our understanding of the factors influencing arbovirus emergence and evolution is the use of experimental studies in the laboratory. Here we review the contributions that laboratory passage and experimental infection studies have made to the field of arbovirus adaptation and evolution. In particular, this review focuses on the areas of evolutionary constraints and mutant swarm dynamics, how experimental results compare to theoretical predictions, the importance of arbovirus ecology in shaping viral swarms, and how current knowledge should guide future questions relevant to understanding arbovirus evolution.2.i.e., fitness trade-offs. The generally accepted theory is that cycling between disparate hosts selects for generalists and, as a consequence, arboviruses sacrifice the ability to be host specialists [Despite the enormous potential for sequence change inherent in RNA viruses, the consensus sequences of most arboviruses have remained highly genetically conserved in nature –33. Thiscialists ,42. Speccialists .in vitro experimental evolution studies. These studies, although lacking the complexity of natural in vivo systems, have been useful tools in beginning to define the selective pressures acting on arboviruses in a simpler setting.Although these concepts are generally accepted, experimental studies have provided mixed results in testing the hypotheses that (a) significant constraints on genetic change of arboviruses result from host cycling, and (b) arboviruses are subject to significant fitness trade-offs as a consequence of host cycling. Here we review the contributions of such studies, beginning with 2.1.Flavivirus consists of more than 70 species, but the virus which undoubtedly has had the most widespread impact on public health, with annual worldwide infections approaching 100 million, is DENV [Aedes albopictus, C6/36) cell culture, and only modest consensus change occurred with sequential passage in mammalian  cell culture [Ae. aegypti mosquitoes and humans also found generally less sequence variation in mosquito-derived isolates [in vitro passage studies evaluated the mutant spectra of experimentally passed strains, a step which is crucial for a comprehensive evaluation of genetic change. In fact, similar passage studies with WNV and SLEV [et al. 2009 DENV study, these studies demonstrated that both WNV and SLEV are capable of significant host-specific adaptation with sequential passage in mosquito cells, yet this seemed to come at little cost in the ‘bypassed’ vertebrate host. Taken together, results from in vitro flavivirus studies do not support the idea that limitations on fixed, consensus change result from cycling alone, nor do they generally support the existence of significant fitness trade-offs resulting from host cycling.The flavivirus genome is single-stranded, positive sense RNA which is approximately 11 kb in length with a single long open reading frame . The gen is DENV . A previ culture , but it  culture . Both DEisolates . Althougand SLEV  demonstrand SLEV . Similar2.2.Alphavirus and many of the experimental evolution studies have focused on important pathogens within this genus [, in vitro passage studies with alphaviruses provide somewhat contradictory results regarding the extent of both fitness trade-offs and evolutionary constraints. A study with EEEV in which virus was passaged sequentially in either vertebrate (BHK) or invertebrate (C6/36) cells, or in alternate hosts, reported that fitness increases were measured in cell lines used for sequential passage and fitness losses were generally seen in bypassed cells [in vitro passage studies with alphaviruses demonstrate that host specialization through sequential passage often result in fitness costs in the bypassed host, and that host cycling may dampen the rate of consensus genetic change; yet these studies also show that host specialization without significant fitness trade-offs is at times attainable through cycling.The majority of togaviruses are mosquito-borne viruses in the genus is genus . The alped cells . Despiteed cells . Althouged cells . In this2.3.VSV, a positive sense, single-stranded RNA virus with 5 distinct genes (ORFs), is the most studied arbovirus in the field of experimental evolution; ironically VSV may not be highly representative of arboviruses in general. Unlike the mosquito-borne flaviviruses and alphaviruses already discussed, which generally have a narrow vector range, a broad range of vectors and modes of transmission have been implicated for VSV. Sandflies, as well as biting midges and mosquitoes play dominant roles in VSV maintenance and transmission ,55, and et al. [et al. [in vitro passage and subsequent evaluation of fitness changes. Novella et al. [vs. acute) in shaping arbovirus adaptation. These results supported the concept of fitness trade-offs with host-specific adaptation. In work by Turner and Elena [versus replicative strategy. A follow-up study investigated this concept and demonstrated that the persistent phase of the cycle (invertebrate) is the dominant evolutionary force and that trade-offs are dependent on strategy and not necessarily host cell type for VSV in vitro [Studies by Holland and colleagues with VSV , and subet al.  and Clar [et al. , was thea et al.  demonstrnd Elena , fitnessnd Elena . In contnd Elena ,53, the in vitro . The idein vitro . Taken t2.4.in vitro passage studies. The appropriateness of many factors including multiplicities of infection, temperatures, number of passages, length of individual passages, measures of viral fitness, and source of the passed virus strains, is not always clear, yet slight variations in these factors may have profound effects on outcomes. Additionally, studies with both alphaviruses [in vitro. These specific examples demonstrate the general fact that in vitro systems are often inapt representatives of natural environments and that experimental passage studies which utilize relevant in vivo systems more closely mimicking natural environments are needed. In 1975, Taylor and Marshall demonstrated that RRV rapidly evolved to increased virulence when sequentially passed in mice; however, alternate passage between Ae. aegypti mosquitoes and mice constrained changes in virulence [in vivo evolution studies have been generally lacking, yet recent work with flaviviruses WNV and SLEV [in vitro findings in relevant in vivo hosts. Sequential passage of VEEV in vertebrates (mice or hamsters) or Ae. aegypti mosquitoes, led to specialized viruses in each host, whereas alternating passage did not result in fitness gains in either host, supporting the idea that cycling constrains host-specific adaptation. Although in this study the presence of potentially important mutant variants was not evaluated, consensus genetic change associated with host-specific adaptations were modest and no greater in number than changes identified in virus subjected to alternate passage. While this demonstrates the ability for further host specialization, these results do not support the idea that rate of evolutionary change is constrained by host cycling. Experimental passage of WNV in Cx. pipiens mosquitoes revealed the capacity for WNV to adapt further to this host, yet no measurable cost was demonstrated in terms of replicative ability in chickens [Cx. pipiens following passage by inoculation. Since release from host alteration does not lead to further adaptation in this study, it suggests, unlike VEEV and WNV work, that SLEV adaptation to mosquitoes in nature may not be significantly hampered by host cycling. These studies also demonstrate that significant adaptation to avian hosts already exists, but some gains in terms of infectivity were possible. An important caveat to WNV and SLEV studies is the use of intrathoracic inoculation rather than bloodfeeding for mosquito passage. Infection, replication, and dissemination from the mosquito midgut may require variants different from those selected for infection of and replication in parenteral tissues; yet to address this experimentally is difficult, as viral titers generally are not sufficiently high to infect a large proportion of the mosquitoes via bloodfeeding without intermediate amplification. This problem was overcome with VEEV by the pooling of mosquitoes [in vivo studies, they provide a much better representation of the complexity of the selective pressures to which arboviruses in nature are subject than do in vitro studies. The fact that even this limited body of work provides results that are not wholly in agreement demonstrates that it may not be possible to use a broad brush to generalize the mechanisms by which arboviral hosts shape the viral population.The fact that some studies, even with the same virus, yield different results points to the importance of the experimental conditions in the various aviruses  and flavaviruses  demonstrirulence . Since tand SLEV –74 and tchickens . Similar2.5.i.e., the idea that cycling should always result in suboptimal adaptation in each host. Arboviruses in the lab and in nature undoubtedly have the capacity to achieve high levels of adaptation to both host environments in spite of cycling; and host-specific adaptations often carry no cost in alternate environments. Although some constraints on host specific adaptation certainly exist, they are often subtle and are species-dependent. This is not surprising since arboviruses differ not only in host utilization but also in genome organization, rates of recombination, breadth of mutant swarms, mechanisms of transmission, and mechanisms of seasonal survival . A complete understanding of how such factors shape arbovirus populations is crucial to understanding arbovirus evolution and epidemiology. Beyond species specific differences, one also must look deeper at gene specific differences. Studies with VSV demonstrate that changes in particular regions result in antagonistic pleiotropy in divergent hosts whereas other mutations may be neutral or co-adaptive in other hosts [Although variability exists between the results of arboviral passage studies completed thus far, there are general conclusions pertaining to host adaptation, viral fitness and viral evolution that are broadly supported. In regards to both the genetic and phenotypic consequences of host cycling, studies by in large refute the inevitability of fitness trade-offs, er hosts ,76. The er hosts . This prCulex mosquitoes [Ae. albopictus mosquitoes [Although it is clear that rates of genetic change in nature are generally low relative to their potential, results from experimental evolution studies as a whole do not support the hypothesis that this slow accumulation of change is a result of host cycling alone. In fact, most studies have demonstrated the same modest accumulation of fixed consensus change occurs with sequential passage and that selective pressure in individual hosts, rather than host alternation, is more likely responsible for the slow rates of evolution in nature. The main caveat to this conclusion is that the majority of these studies consider only consensus level change. Furthermore, modest change in terms of numbers of mutations is not always synonymous with the phenotypic impact of change. Single substitutions can have profound effects on replicative ability and/or infectivity in particular hosts; this has not only been observed experimentally, but also in nature. Genotypes of VEEV associated with outbreaks have been shown to have single mutations in the E2 gene responsible for increased vector competence  or equinsquitoes  and thissquitoes –84, incrsquitoes –87. Thes3.Arboviruses often exist as a collection of variable genomes within a host. This mixed population of genomic variants, collectively referred to as the mutant swarm or mutant spectrum, is the result of a rapid replication rate combined with the error prone nature of viral RdRps. Although many refer to this swarm as a ‘quasispecies’ structure, the origin of the term quasispecies  describe3.1.One clear advantage diverse mutant populations possess is phenotypic plasticity and adaptability to new and dynamic environments. It seems that this adaptability may indeed be required for all RNA viruses, as recent studies with poliovirus have demonstrated that high fidelity mutants that are constrained in their capacity for exploration of sequence space are often highly attenuated, and therefore, promising vaccine candidates –97. ConvPhenotypic plasticity is a characteristic of highly diverse populations, which is particularly important for arboviruses that replicate in both highly divergent hosts and diverse tissues within each host. Extreme fitness losses of VSV in the vertebrate environment resulting from passage in sandfly cells can be almost completely reversed with a single passage in BHK cells, a result that plainly demonstrates the ability of the viral mutant swarm to maintain variants in a population which have proven useful in the past . This abCulex mosquitoes [Cx. quinquefasciatus mosquitoes [Selective pressures that arboviruses encounter in vertebrate and invertebrate systems are undoubtedly very different. In contrast to what has been shown for DENV-3 , intrahosquitoes . In a susquitoes . While isquitoes . The RNAsquitoes –121. It squitoes ,123. A m3.2.In conjunction with the benefit of adaptability which may result from increases in mutant swarm breadth, a role for minority variants in viral fitness is also well defined ,113,124.In vitro evidence of strain complementation at high MOIs exists for VSV [Intriguing evidence exists for cooperative interactions other than recombination among individual virus strains, specifically via complementation. A defective strain of DENV-1 containing a stop codon in the envelope gene was found to be maintained in both humans and mosquitoes in Myanmar over a period of at least 18 months . Phyloge for VSV , in whic for VSV  and othe for VSV . In fact for VSV .et al. [There is limited knowledge about the distribution of fitness values within a given viral swarm at any one given time as a consequence of the dynamic character of a mutant spectrum in nature. The majority of variants within a high fitness population of VSV were found to have fitness values that were on average lower than the population as a whole . This iset al. . Whether3.3.Defining the role of bottlenecks in shaping the arbovirus mutant spectrum is crucial to understanding arbovirus evolution. Both theoretical and experimental studies demonstrate that RNA viruses are particularly vulnerable to significant fitness losses from frequent and tight bottlenecks (Muller’s ratchet) due to their inherent propensity to produce deleterious variants ,145,146.Cx. pipiens, accumulation of genetic diversity was noted during passage by inoculation when whole bodies were analyzed [6 plaque forming units of virus [For arboviruses that utilize mosquito vectors, bottlenecks will occur upon infection of midgut cells, egress from the midgut, infection of parenteral tissues including the salivary glands, and subsequent egress into the salivary secretion during transmission to vertebrate hosts –151. Witanalyzed , yet wheanalyzed . By bypaof virus , it remaBuggy Creek virus, can survive for long periods without a vertebrate host and have been found to have a high frequency of infection during winters in the Great Plains in the United States [Aedes triseriatus mosquitoes are capable of transmitting LACV to over 80% of their progeny and venereal transmission also occurs [Cx. annulirstris but in the eggs of Ae. vigilax [Potentially the most significant of all the bottlenecks to which arboviruses are subject are those imposed on viruses which require mechanisms to survive seasonal interruptions in transmission cycles. Phylogenetic studies indicate that most arboviruses are maintained locally, yet the mechanisms for this seasonal maintenance are variable. Some insect vectors may remain persistently infected through winter or other breaks in transmission. For example, ticks infected with Langat virus are still capable of transmitting virus after more than three years ,155. Swad States –160 whicd States ,163). Ind States ,165. Somo occurs . Mechani vigilax ,168, dif vigilax , yet theUltimately, a virus’ potential to survive and persist following naturally occurring genetic bottlenecks is important to its potential for host range shifts and expansion, and likely has major implications for predicting how viruses will evolve in terms of human susceptibility and pathogenesis. For example, the North American and South American strains of the alphaviruses EEEV differ greatly in their ability to cause neuroinvasive disease in humans . These d4.Arboviruses are bound by their need to both infect and cycle between vertebrate and arthropod hosts. It is because of this need that all arboviruses are required to either be generalists or possess some means of phenotypic plasticity. Despite these shared requirements, attempting to generalize findings on arbovirus evolution fails to recognize the enormous diversity in viral genomes and their replication strategies, hosts, and ecology that exists among these viruses.in vitro systems have been highly informative in studying basic concepts, the natural hosts ultimately are required to understand mechanisms of viral adaptation and evolution. Although such in vivo experimental studies are beginning to be undertaken, significant expansion of such studies with a focus on host- and virus-specific differences will help to elucidate the unique interactions that shape the evolution of these complex systems.In conjunction with the outlined need for a more accurate definition of the role of both minority variants and the arboviral swarm in general, a more complete understanding of how these laboratory defined mechanisms translate to functional and, therefore, evolutionary consequences in natural systems is needed. While Phylogenetic studies to date rely exclusively on compilations of consensus sequences from multiple virus isolates. Such studies are highly informative; however, there is a need for large scale evaluation of intrahost genetic diversity both spatially and temporally in nature in order to fully understand the complexity of evolutionary history, the influence of seasonal and within host bottlenecks, and the potential for both phenotypic change and host expansions.


To the Editor: We report 2 cases of possible recurrent laboratory-confirmed infection with pandemic (H1N1) 2009 virus in Israel. Patient 1, a 24-year-old man, had Noonan syndrome  2009 virus. On November 22, the man was hospitalized again for dyspnea and fever. The RT-PCR result from a nasopharyngeal sample collected at admission was positive. Hemagglutination-inhibition assay demonstrated a high titer (320) of serum antibody against pandemic (H1N1) 2009 virus in a blood sample taken at admission. The patient took oseltamivir for 5 days, and his condition markedly improved. Result of a repeat RT-PCR at discharge was negative.An identical neuraminidase gene sequence was detected during both illness episodes (August and November). The specimens were also tested with an experimental RT-PCR assay for rapid detection of the oseltamivir resistance mutation H275Y on the pandemic neuraminidase gene  2009 virus was positive on August 14. A second course of oseltamivir was administered for 10 days with the dosage adjusted for age and doubled from that of the previous regimen. Further testing with the experimental rapid RT-PCR indicated the viral strain had the oseltamivir resistance mutation. On September 14, RT-PCR was negative.On December 11, the boy was again hospitalized because of respiratory distress and high-grade fever. On December 14, RT-PCR was positive for pandemic (H1N1) 2009 virus, and a 5-day regimen of oseltamivir was started. Another specimen taken the same day was negative. A high serum antibody titer (320) to pandemic (H1N1) 2009 virus was measured by hemagglutination-inhibition assay on December 16; no oseltamivir resistance mutation was found. Additional laboratory testing included a complete panel for respiratory viruses, which was negative for human metapneumovirus; respiratory syncytial virus; adenovirus; seasonal influenza virus types A and B; and parainfluenza virus types 1, 2, and 3.,These 2 cases of possible recurrent pandemic (H1N1) 2009 infection demonstrated a wide interval between illness episodes. Neither patient had accompanying immunodeficiency, and both had antibody titers far beyond the accepted seroprotective threshold for influenza (www.sii.org.il/20-en/SII_EN.aspx). Furthermore, no other respiratory virus was found by laboratory testing at that time. The patient was infected with an oseltamivir-resistant pandemic (H1N1) 2009 virus during the first illness episode and with an oseltamivir-sensitive virus during the second episode and had 2 RT-PCRs with negative results between the episodes.Virus clearance was not laboratory confirmed for patient 1 after the first episode because no samples were taken after hospital discharge. Patient 2 had both positive and negative RT-PCRs for pandemic (H1N1) 2009 virus  from samThe novel pandemic influenza virus may be able to reinfect certain chronically ill persons. Caregivers should be aware of this trait when considering the differential diagnosis of influenza-like illness in a patient with a documented, and even treated, pandemic (H1N1) 2009 infection.


Adults with certain comorbid conditions have a higher risk of pneumonia than the overall population. If treatment of pneumonia is more costly in certain predictable situations, this would affect the value proposition of populations for pneumonia prevention. We estimate the economic impact of community-acquired pneumonia (CAP) for adults with asthma, diabetes, chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF) in a large U.S. commercially-insured working age population.Data sources consisted of 2003 through 2007 Thomson Reuters MarketScan Commercial Claims and Encounters and Thomson Reuters Health Productivity and Management (HPM) databases. Pneumonia episodes and selected comorbidities were identified by ICD-9-CM diagnosis codes. By propensity score matching, controls were identified for pneumonia patients. Excess direct medical costs and excess productivity cost were estimated by generalized linear models (GLM).We identified 402,831 patients with CAP between 2003 through 2007, with 25,560, 32,677, 16,343, and 5,062 episodes occurring in patients with asthma, diabetes, COPD and CHF, respectively. Mean excess costs  of CAP were $14,429 (SE=44) overall. Mean excess costs by comorbidity subgroup were lowest for asthma ), followed by diabetes ) and COPD ); mean excess costs were highest for patients with CHF ). On average, indirect costs comprised 21% of total excess costs, ranging from 8% for CHF patients to 27% for COPD patients.Compared to patients without asthma, diabetes, COPD, or CHF, the excess cost of CAP is nearly twice as high for patients with diabetes and COPD and nearly three times as high for patients with CHF. Indirect costs made up a significant but varying portion of excess CAP costs. Returns on prevention of pneumonia would therefore be higher in adults with these comorbidities. Community-acquired pneumonia (CAP) ranks among the top causes of death and hospitalization and it is a major driver of health care utilization and cost. Annually, there are 2 to 3 million CAP cases in the US resulting in half a million hospitalizations, 10 million physician visits, and 45,000 deaths , the cosWe estimate the total excess cost burden of CAP as the sum of medical costs and productivity losses (work absence and short-term disability) in a large cohort of commercially insured working-age adults throughout the United States for the most common comorbidities among individuals with pneumonia: asthma, diabetes, COPD, and CHF ,8. In adThis project utilized two Thomson Reuters’ research proprietary databases. For estimations of medical cost, the study is based on medical and outpatient prescription drug claims of employees and dependents in the MarketScan Commercial Claims  and Encounters database, which included 34 million covered lives in 2008. For estimations of productivity cost, the study used workplace absenteeism and short-term disability data from the Health Productivity and Management (HPM) database. The HPM database is constructed from administrative payroll system data that includes missed work, short-term disability, and worker’s compensation from some of the same employers that have provided health care claims. HPM data has been widely used in academic publications -16. HPM Pneumonia episodes were identified by ICD-9-CM diagnosis codes (480.xx-486.xx) in (1) a principal or non-principal diagnosis on an outpatient claim or (2) a principal diagnosis on an inpatient claim. An index date for pneumonia was defined as the date of its first diagnosis without any similar diagnosis  in the prior six-months.In order to obtain comparable controls, a control group was created from a pool of controls using a nearest neighbor propensity score 1:3 case:control match without replacement. The pool of potential controls was constructed from all individuals without pneumonia. To allow pre-index group comparisons and identification of selected comorbidities, control patients had a randomly assigned index date and were required to also have 6 months of continuous medical and prescription coverage preceding the index date. The propensity score of the presence of a pneumonia diagnosis was based on a logistic regression using the following covariates: index year, quarter, age, gender, geographic region, plan capitation status, industry, length of follow-up, baseline Deyo Charlson Comorbidity Index, and baseline comorbid conditions, including asthma, diabetes, cancer, cardiovascular disease, CHF, COPD, and HIV. We used this same technique to obtain controls for the subset of patients with work absence and short-term disability data.There was no minimum enrollment time requirement following the index dates for cases and controls. The maximum follow-up time was 12 months from the index date, depending on enrollees' data availability.As the focus of this study was community-acquired pneumonia (CAP), we excluded from the cost analysis any health-care associated or hospital-acquired pneumonia, which was defined as principal diagnosis on an inpatient claim that had evidence of an acute-inpatient or long-term care stay in the previous 30 days.The outcome variables include three components of medical costs: inpatient, outpatient, and pharmacy; and two components of productivity costs: absenteeism costs and short-term disability costs.Medical costs were defined as total gross payments to a provider, including deductibles, copayments, and coordination of benefits. Costs were tabulated separately for inpatient medical, outpatient medical, and outpatient pharmacy. The costs of emergency room visits that led to an inpatient visit were classified as inpatient medical; all others were classified as outpatient medical. Pharmaceutical costs equaled the actual reimbursement to the pharmacy, including the drug’s administrative dispensing fee, ingredient cost, patient portion of costs, and sales tax. A payment proxy based on procedure code and region of care by the data provider was estimated for expenditures for services provided under capitation arrangements. All medical costs were adjusted to 2008 dollars using the Medical Care-Specific Consumer Price Index.To estimate the cost of absenteeism, the number of employer-recorded days of absence from work was multiplied by a wage constant of $41/hour. This wage is based on a previous benchmarking study of health and productivity-related costs from a sample of 32 companies across the US  and adjuOur strategy to estimate excess costs was to use a multivariate model with cases and controls with excess costs defined as the adjusted difference in costs between cases and controls. By including a control variable for number of months of data availability following the index date, we annualize costs through a post-estimation prediction under the assumption that all subjects are followed for 12 months.Based on a comprehensive comparison of estimation methods in earlier analyses on costs of pneumonia , we estiSeparate models were estimated for all subjects and for each of the selected comorbidity subgroups . We note these comorbidity subgroups are not mutually exclusive. A separate subgroup was defined by the absence of all four of these comorbidities. Code lists are available from the authors upon request.We identified 402,831 patients with CAP from MarketScan Commercial Claims  and Encounters database. The overall demographic characteristics and pre-index period clinical characteristics are shown in Table Demographic characteristics were similar between CAP patients and their 1,425,045 matched controls as the standardized differences were typically in the 1-3% range. The highest standardized differences were COPD at 7.29, comprehensive insurance at 7.39, and pre-period outpatient costs with standardized differences above 10; multivariate models were used to control for these remaining imbalances.Annualized adjusted excess total medical costs of a case of CAP were estimated at $11,395 (SE=$42) across all patients, as determined by the difference between estimated costs for patients with CAP ) and the control patients ). These estimates come from costs defined in 2008 dollars as total gross payments to a provider, including deductibles, copayments, and coordination of benefits.Excess medical costs of CAP among patients with COPD, diabetes, and CHF are higher than the average excess medical costs of CAP. Pneumonia is most costly among CHF patients at $31,593 (SE=$531). Excess costs are also substantially higher for patients with COPD ) and diabetes ). Among the comorbidities of interest, CAP with asthma had the lowest total excess medical costs at $10,158 (SE=$109). All of these costs were higher than the excess costs of CAP for patients without CHF, COPD, diabetes, or asthma ).The detail of medical costs by each cost component sheds light on the drivers of higher excess medical costs among the comorbidity subgroups. As shown in Table As shown in Table Table The objective of this study was to estimate, for selected comorbidities, the excess health care costs and employer productivity costs of working age adults with CAP. The excess costs per case of CAP in the working-age adult population was $14,429 overall, but could be nearly 3 times as high among patients with CHF and around 75% higher among patients with COPD and diabetes. When considering the reduced cost burden from prevention of CAP, it is not sufficient to consider the risks of acquiring pneumonia, the fact that the cost per case can vary among different risk groups must be taken into account. As a result, we have provided key estimates of the burden of disease among key comorbidity risk groups.The cost per CAP patient is highest for patients with CHF  and is substantially higher than the cost of CAP for patients with other conditions such as COPD $23,439), diabetes $21,395) and asthma , and particularly those without any of these selected comorbidities . The higher costs, particularly for CHF and even for COPD may be explained by the clinical evidence which suggests that the pre-existing condition has a detrimental effect on pneumonia prognosis ,21, but ,395 and , diabeteWith no other comorbidity-specific estimates of pneumonia costs, these results cannot be directly compared to other studies in the literature, but the overall estimates are in line with the literature. For example, our work suggests that productivity costs are about 20% of total burden. The one previous analysis that included productivity costs suggested that they represent 29% of total cost . Colice A recent European study of patients with CAP reported no difference in median hospitalization costs between patients with and without diabetes, CHF, or COPD . The anaThe analyses described herein have limitations. While the MarketScan Research Databases represent a wide variety of nationally-representative employed patients and their dependents, it is not a random sample. As a result, the study results may not be generalizable to groups beyond the commercially insured, such as Medicaid beneficiaries or unemployed patients. Misclassification of pneumonia due to inaccurate or absent diagnosis coding is possible when analyzing claims data and basing the pneumonia diagnosis on ICD-9 codes alone and not on review of medical records or X-ray results. For example, it is possible that some mild cases were coded as “respiratory infection” and a few severe cases were coded as “sepsis”. Likewise, the intention of this analysis was to describe the burden of CAP separate from HAP; our ability to differentiate CAP from HAP may be limited by information available in an administrative claims database. Including HAP cases in our cohort of CAP patients could lead to an overestimation of the burden of CAP. However, to reduce the possibility of including HAP, we excluded from the analysis any diagnosis of pneumonia in an inpatient claim that was based on a secondary diagnosis or even in a principal diagnosis if there was evidence of an inpatient or long-term care stay in the previous 30 days. Productivity costs are estimated from the employer perspective and include only missed work time, not work time completed at a reduced productivity level. If illness affects labor force participation or has an effect on long-term disability, these productivity costs will not be captured. Finally, our method of estimating “excess costs” depends on the degree to which our matched control group is an accurate representation of the counterfactual of what the pneumonia patients would have experienced had they not contracted pneumonia. This assumption is not testable so our results must be interpreted in light of this uncertainty.In summary, we found that CAP is associated with substantial excess medical and productivity costs that can vary substantially by comorbidity group. Excess costs per case are highest for patients with a comorbidity of CHF and also elevated for patients with diabetes and COPD. Returns on prevention of pneumonia would therefore be higher in adults with these comorbidities than in adults without any of the selected comorbidities.CAP: Community Acquired Pneumonia; HAP: Hospital Acquired Pneumonia; COPD: chronic obstructive pulmonary disease; CHF: Congestive Heart Failure; SE: Standard Error; SD: Standard Deviation; HPM: Thomson Reuters Health Productivity and Management database; CCI: Charlson Comorbidity Index.Dr. Polsky has received consulting fees from GlaxoSmithKline. Dr. Suaya is employed by the GlaxoSmithKline group of companies. At the time of this study, Suaya worked at GlaxoSmithKline Vaccines. Dr. Bonafede is a employee of Thompson Reuters.All three authors have contributed to the conception, design, analysis, and interpretation of the data; the drafting and revising of the manuscript for important intellectual content; and have given final approval.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/379/prepub


Bodhi beads are a Buddhist prayer item made from seeds. Bodhi beads have a large and emerging market in China, and demand for the beads has particularly increased in Buddhism regions, especially Tibet. Many people have started to focus on and collect Bodhi beads and to develop a Bodhi bead culture. But no research has examined the source plants of Bodhi beads. Therefore, ethnobotanical surveys were conducted in six provinces of China to investigate and document Bodhi bead plants. Reasons for the development of Bodhi bead culture were also discussed.Six provinces of China were selected for market surveys. Information was collected using semi-structured interviews, key informant interviews, and participatory observation with traders, tourists, and local residents. Barkhor Street in Lhasa was focused on during market surveys because it is one of the most popular streets in China.Forty-seven species (including 2 varieties) in 19 families and 39 genera represented 52 types of Bodhi beads that were collected. The most popular Bodhi bead plants have a long history and religious significance. Most Bodhi bead plants can be used as medicine or food, and their seeds or fruits are the main elements in these uses. ‘Bodhi seeds’ have been historically used in other countries for making ornaments, especially seeds of the legume family. Many factors helped form Bodhi bead culture in China, but its foundation was in Indian Buddhist culture.As one of the earliest adornment materials, seeds played an important role for human production and life. Complex sources of Bodhi beads have different cultural and historical significance. People bought and collected Bodhi beads to reflect their love and admiration for the plants. Thus, the documentation of Bodhi bead plants can serve as a basis for future investigation of Bodhi bead culture and modern Buddhist culture. Humans use seeds  in many ways. Seeds are colorful, durable, and easy to access, so humans have drilled and strung seeds into necklaces and bracelets for thousands of years. The oldest seed ornaments were discovered in Africa and can be dated to the Middle Stone Age  . MoreovePu Ti Zi in Chinese: Pu Ti means Bodhi tree (Ficus religiosa), and Zi means seed; but Bodhi beads are not made of the seeds of a Bodhi tree (Ficus religiosa). The name Pu Ti Zi first appeared in the early medical text, ‘Compendium of Materia Medica’, which referred to the seeds of Coix lacryma-jobi[Sapindus delavayi and Sapindus tomentosus were called Pu Ti Zi or ‘Bodhi seeds’ in Yunnan [Bodhi beads are Buddhist prayer items that have been traditional tools for counting while reciting a mantra, as prayer beads have been used in other world religions. The Bodhi beads are called ryma-jobi. In addin Yunnan . In modeBodhi beads have an important position in Tibetan Buddhism and are very popular in Tibet (the Xizang Tibetan Autonomous Region). The culture of Bodhi beads spread throughout Tibet and other provinces of China, and there are dozens to hundreds of Bodhi beads with different meanings. Recently, people have begun to wear Bodhi beads as a kind of praying or blessing ornament, in addition to functioning as a prayer bead. The Bodhi bead culture has developed in China in the form of cultural supplies, such as writing brushes and ink sticks. Several ‘Bodhi seeds’ of similar size, shape, and pattern are strung into strings of prayer beads. People frequently touch the beads with their hands to make the beads luster like jade due to secretion from human skin. Moreover, the beads are carefully modified to protect from scarring in inappropriate temperature and humidity surroundings.Ethnobotanists study how plants are used for food and medicine but are also interested in plant adornments because non-mainstream use of plants can reflect relationships between cultures and (uses of) plants in other aspects. Surveys such as that conducted by Armstrong have shown that many botanical jewelries in different places were made by seeds and indicated the high value placed on the seeds ,6. Over Pu Ti Zi and explored the culture values of Bodhi bead plants.Recently, there has been increasing interest in the commercial value of Bodhi beads, and many books about the culture of Bodhi beads have appeared in China. Although the books and related materials focus on the meaning of each kind of Bodhi bead, almost all publications ignore the question of which plant or plants produced the Bodhi seeds. The beads have been commonly described as ‘seeds of rare plants’, and this description has fostered a belief that the value of Bodhi beads is related to the scarcity of their sources and not to their aesthetic and cultural value. To help correct this misunderstanding, we investigated the plant sources of ‘Bodhi seeds’ or Surveys were conducted in local markets and e-commercial platforms of six provinces in China: Tibet, Yunnan, Fujian, Zhejiang, Beijing, and Guangdong. Field work was conducted from September to October 2012 and August to September 2013. Barkhor Street  is a famous street circling Jokhang Temple in Lhasa, Tibet, and was the main local market surveyed. The traditional culture and local lifestyle have been well-preserved for more than 1300 years. We choose this market because of the wide variety of Bodhi beads traded there between vendors, pilgrims, and tourists from Nepal, Bhutan, India, and other places in China. It is the most important commercial center, folk-custom heritage, tourist attraction , and shoBodhi beads samples were purchased from Barkhor Street markets and e-commercial platforms. A detailed inventory and related information for the Bodhi beads were prepared from semi-structured interviews, key informant interviews, and participatory observation with traders, tourists, and local residents. In order to access the popularity of Bodhi beads in Chinese markets, various Bodhi beads in 126 shops of six provinces were studied. The number of shops that each Bodhi bead was sold in was counted for an occurrence frequency (OF) calculation; for example, if N shops sold Bodhi bead type ‘A’ (N ≥ 1), then the occurrence frequency of ‘A’ is OF(A) = N/126 × 100%.All seeds were identified according to morphological characters and geographical origins using standard literatures -12 and cLatania loddigesii – ‘Xian-Zhi’) in IUCN [Dracontomelon macrocarpum – ‘Grimace’ and Cycas revoluta – ‘Buddha-mind’) in the China Species Red List [Caesalpinia (3 identified species). Most seeds of a particular species can produce only one type of Bodhi bead. However, different degrees of processing of Corypha umbraculifera, Daemonorops jenkinsiana, and Elaeis guineensis can be used to make different types of Bodhi bead. The seeds with 2-, 3- or 4-loculed drupes of Ziziphus abyssinica can be used to make ‘Phoenix eye’, ‘Dragon eye’, and ‘Kylin eye’, respectively. Chinese names of most Bodhi beads are according to the morphological characteristics of their respective seeds; for example, the beads called ‘Moon and Stars’ usually have an ivory surface with small holes (moons) and tiny black dots (stars). Seeds of Caesalpinia bonduc are grayish, shiny, ovoid to globose, and look like the moon; therefore, the name of the corresponding Bodhi bead is ‘Moon seed’.Fifty-two types of Bodhi beads were documented Table , made of in IUCN  and two Red List . The majPhytelephas macrocarpa, Caesalpinia major, and Ziziphus abyssinica. Thirty-one species are native to China, with 19 species mainly distributed in tropical and subtropical areas, and the other species widely distributed throughout China. Twelve species have only cultivation types, and most of these species have been domesticated in southwestern and southern China.Three species of Bodhi bead plants do not occur in China: The occurrence frequencies of four types of Bodhi beads reached at least 50%, including ‘King Kong’, ‘Moon and star’, ‘Bodhi root’, and ‘Phoenix eye’ Figure . These wElaeocarpus angustifolius. ‘Two-furrowed King Kong’ is made from the fruit of Elaeocarpus hainanensis. Elaeocarpus plants are distributed in Hainan, Yunnan, Guangxi, Tibet of China, and other parts of Asia, including Nepal, Bhutan, India, Indonesia, and the lowlands of the Himalayas [The most common Bodhi bead, ‘King Kong’, is made from the fruit of imalayas . ‘King Kimalayas , named ‘imalayas . The furimalayas . Five fuimalayas . Normallimalayas .Daemonorops jenkinsiana. The ‘Moon and stars’ name reflects the small holes (moon) and tiny black dots (stars) covering the seed’s surface. This species is mainly distributed in the south of China, India, Nepal, Bhutan and Bangladesh. There was no record of the history of this type of Bodhi bead, but we found many old ‘Moon and stars’ beads in Tibet; we therefore speculated that Tibetan Buddhism was influenced by mainland China’s Buddhism.‘Moon and stars’ is a very popular and traditional Bodhi bead in Chinese Buddhism and is the hard and dense seed of Corypha umbraculifera are used to make ‘Bodhi root’ beads. This species is native to India and Sri Lanka. In India, it is also called ‘vegetable ivory’ and is a traditional tool for carving Buddhist Sutras, such as the famous ‘tale palm’ or ‘tad-patri’ because the leaves are flexible and soft when dry [C. umbraculifera was cultivated in temple gardens in Xishuangbanna of Yunnan Province in China, and over 50,000 volumes of Buddhist Sutras carved on leaves were protected there [C. umbraculifera is regarded as one of the iconic Buddhist plants in Asia.Seeds of when dry -32. Becawhen dry , C. umbred there . In addied there . TherefoZiziphus abyssinica and Ziziphus jujuba var. spinosa, respectively. The name ‘Phoenix eye’ refers to the eye-like shape that appears on the hard endocarp. Z. abyssinica is native to India and is not cultivated in China. Both were used to make adornments in India [Ziziphus has been mentioned more than one time in the Ramayana and the Mahabharata [Z. spina-christi) is the only tree that could be regarded as a holy tree in Islam [Ziziphus are also held sacred by many other religious persons, such as some Druze, Muslims, and Christians.‘Phoenix eye’ and ‘Small phoenix eye’ are made from the fruits of in India ,36. Ziziabharata . The trein Islam . SpeciesCanarium spp.) have a long history as accessories in China. Drupes of Chinese black olives (Canarium pimela) are the main material for carving and playing and are produced mainly in the Chinese provinces of Guangdong and Fujian. ‘Olive-stone carving’ prospered in the Ming to Qing (1368–1911) dynasties, although it might have originated earlier. The most classical ‘olive-stone carving’ is the ‘Olive nut boat.’ Playing with walnuts is a traditional Chinese practice with its origin in traditional Chinese medicine therapies: the Chinese consider the bumps and sharp edges of a walnut to promote blood circulation when playing with walnuts in one’s hands [Juglans species in China can be used for playing with walnuts, including Juglans regia.Chinese olives  are also used as medicine. The seeds or fruits of most species are the main medicinal parts. Some species can be used to treat heart disease, dyspepsia, hypertension, rheumatism, and other ailments. Except for eumatism . Althougmedicine . Twenty th China . Leaves nd so on . Sixteenhe world .As one of the most popular Buddhist supplies, Bodhi beads are being paid more attention and becoming the target of many collectors. However, the sources of Bodhi beads have been ignored.The purpose of the current study was to give an account of Bodhi bead plants and the reasons for the widespread use of Bodhi beads in China. Seeds of 47 species (2 varieties) were recorded to be used for making 52 types of Bodhi bead. Some of these seeds were also used in other countries as necklaces or strings of prayer beads. They might be the oldest materials used for ornaments. Twelve of them were traditional Indian beads, especially rudraksha seeds. Together with other items, they were the earliest prayer beads in Indian Buddhism. We also found that all of the most popular Bodhi bead plants had a close historical relationship with religion. Bodhi bead plants have multiple uses in China. Besides aesthetic functions, they can be used as food, medicine, timber, and plant oil. These seeds and other parts of plants played a prominent role in the lives of humans for thousands of years. Bodhi beads might be the byproducts of plants, and use of seeds for Bodhi beads might have increased the conservation value of these plants and helped biodiversity conservation in China. The evidence from this study suggests that Buddhism culture combined with Chinese collection culture resulted in modern Bodhi bead culture and that commercial interest in Bodhi beads promotes the spread of Bodhi bead culture.The authors declare that they have no competing interests.FL, JL, JZ, and CL participated in the field survey and conceived of the study. FL, CL, and BL identified all Bodhi bead plants. FL coded all the data and wrote the first draft of the manuscript. All authors contributed to the interpretation of results and contributed to the final manuscript. All authors read and approved the final manuscript.Chunlin Long is a professor at the College of Life and Environmental Sciences, Minzu University of China, Beijing, and research professor at the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming. His research focuses on ethnobotany and ethnomedicine, biodiversity, and plant genetic resources. Feifei Li is a postdoctoral research fellow at the College of Life and Environmental Sciences, Minzu University of China, Beijing. Jianqin Li is an associate professor at the Faculty of Forestry, Southwest Forestry University, Kunming, and Ph.D. candidate at the College of Life and Environmental Sciences, Minzu University of China, Beijing. Bo Liu is a lecturer at the College of Life and Environmental Sciences, Minzu University of China, Beijing. Jingxian Zhuo is a master degree student at the College of Agronomy and Biotechnology, Yunnan Agricultural University, and the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming.