Asthma is associated with enormous healthcare expenditures that include both direct and indirect costs. It is also associated with the loss of future potential earnings related to both morbidity and mortality. The objective of the study is to determine the burden of disease costs associated with asthma.We performed a systematic search of MEDLINE, EMBASE, CINAHL, CDSR, OHE-HEED, and Web of Science Databases between 1966 and 2008.Sixty-eight studies met the inclusion criteria. Hospitalization and medications were found to be the most important cost driver of direct costs. Work and school loss accounted for the greatest percentage of indirect costs. The cost of asthma was correlated with comorbidities, age, and disease severity.Despite the availability of effective preventive therapy, costs associated with asthma are increasing. Strategies including education of patients and physicians, and regular follow-up are required to reduce the economic burden of asthma. Asthma is an inflammatory disorder of the lungs that affects people of all ages and is a significant source of morbidity and mortality worldwide ,2. ApproThe economic costs associated with asthma are estimated to rank as one of the highest among chronic diseases due to the significant healthcare utilization associated with this condition. Numerous studies have been published evaluating the economic burden of asthma on society and individuals. However, a systematic review of the financial impact of asthma has not yet been performed -8. The gA systematic review was conducted to identify English language articles published between 1966 and January 2008 in which the costs of asthma were included. Only studies reported in English (as there was no translator available) and published literature, were included.The following electronic databases were searched using MEDLINE, EMBASE, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Database of Systematic Reviews (CDSR), Health Economic Evaluation Database (OHE-HEED), and Web of Science. Search terms were investigated, including: "asthma", "direct service costs", "cost of illness", "cost- benefit analysis" and "health care costs". Duplicate citations were identified and removed using RefWorks online bibliographic management tool.The titles and abstracts of all publications identified through the primary literature search were independently reviewed by two investigators. The inclusion and exclusion criteria used for study selection are outlined in Table The types of cost related to illness were divided into direct and indirect costs and were defined as follows: direct costs related to direct health service costs and included alternative treatment/medications, physiotherapy/chiropractic, peak flow meters, primary care consultations, paid help for housekeeping, hospital emergency and outpatient attendance, ambulance and other transportation, and hospital admissions. Indirect costs were those applicable to individual patients, their families, and lost opportunities for work or education. The total cost is an aggregate of both direct and indirect costs. For cost data from the United States (US), costs were converted to 2008 US dollars using the medical care component of the consumer price index from the US Bureau of Labor Statistics.For cost data from non-US countries, figures were first converted to 2008 currency values using that country's consumer price index. Figures were then converted to 2008 US dollars, using currency exchange rates of Australian dollar (AUD)$1.00 = United States dollar (USD)$0.637, Canadian dollar (CAD) $1.00 = USD$0.806, (Euro currency code) EUR€1.00 = USD$1.281, and Great Britain pound (GBP)£1.00 = USD$1.427. If the year of the cost data was not reported, it was assumed to be the publication year of the article. In all cases, both the original cost figures provided in the publications as well as the equivalent costs in 2008 US dollars were reported.A standardized data abstraction form was used for all publications included in this study. The data abstracted included the following information: manuscript authors, year of publication, study design and duration, patient characteristics , method of cost calculation, direct costs, productivity (indirect) costs, and total costs. Discrepancies in data abstraction were resolved by consensus.The quality of the economic studies was assessed using a customized version of the Drummond and Jefferson criteria Table  9]. Qua. Qua9]. Due to the heterogeneity in cost analysis and reporting across the studies, a quantitative meta-analysis to aggregate cost data could not be performed. Specifically, the resources used to derive direct healthcare and productivity costs varied substantially between the studies, as did the type of currency . In the absence of the ability to complete a meta-analysis, we opted to complete a qualitative analysis. There were not sufficient homogeneity in terms of participants, interventions, and the way outcomes were defined and measured, to provide a meaningful summary for considering the meta-analysis.The primary literature search identified 2,976 citations. After removing duplicate citations we were left with 2,073 unique citations for screening. The manual screening of all 2,073 titles and abstracts yielded 307 articles that contained primary clinical data evaluating the cost of asthma. Of the 307 full articles retrieved and reviewed by the investigators, 68 met the inclusion criteria , cross- sectional studies (n = 22), and case-control studies (n = 3). Of the 68 studies, only six completed sensitivity analysis -15. Comm, cross- Of the 68 studies identified in the literature review, twenty-three used data derived from the US, twenty-five from European countries, eight from East Asia and the Pacific regions, five from Canada, and seven were from other countries. Twenty-eight studies reported mean or median per patient direct and indirect costs of asthma, thirteen only reported the total direct costs of asthma, and fifteen studies reported annual or total asthma related healthcare expenditures. The economic impact in five studies was based on charge data (not costs) ,44-47.The studies presented in this review used several methods of cost calculation. The most common method was to abstract mean patient resource utilization, such as the number of clinic visits, hospitalizations and procedures, through chart reviews or insurance databases, and to combine these data with the mean unit costs of each resource, derived from local or national accounting databases. An alternative method was to follow a cohort of subjects via a cost-accounting system to capture the charges or costs incurred over time in the management of asthma symptoms.Cost of asthma includes the components of both direct and indirect costs. Direct costs include inpatient care, emergency visits, physician visits, nursing services, ambulance use, drugs and devices, blood and diagnostic tests, research, and education. Indirect costs or morbidity costs include school days lost, traveling, waiting time, and lost productivity for the caretaker of asthmatic children. Direct costs have been shown to exceed indirect costs, and the major components of the direct medical costs found were pharmacological expenditures and hospital admissions. An increase in direct medical costs can potentially lead to a reduced total cost of care if it results in a disproportionately greater reduction in indirect costs due to improved clinical outcomes. In our systematic review, nine studies have found that the direct cost of asthma accounted for the greatest part 53–100%) of the overall cost ,42,47-50–100% of In this systematic review, the largest amount of direct costs found were those allocated to in-patient hospitalization, accounting for 52 to 86% of the overall asthma-related costs in seven studies, ,42,53-55There are several factors that can contribute to higher total hospital costs. Two studies found the following variables correlated significantly with higher hospital costs: older patients, significant comorbidities, intensive care unit (ICU) admission, increasing severity, and prolonged length of stay. In both studies, a small proportion of asthmatics accounted for a large proportion of the total hospital costs ,56. A siStanford and associates reported resources such as nursing care, respiratory therapy, and ED-specific supplies along with equipment use and physician fees to account for the majority of hospital costs of asthma; 44%, 11% and 53% of the costs, respectively . The resA patient's characteristics can also affect the cost of hospitalization. In a study conducted in France, the cost of a hospital stay was compared between a well-managed asthmatic group and a poorly managed one before hospitalization based upon guidelines (11 criteria judged by experts). The poorly managed group was older, tended to include more smokers, spent half as much time in ambulatory care, and had a shorter length of hospitalization. The cost of a hospital stay was found to be 1.72 times higher in the better managed group, due mainly to the differences in the length of the hospital stay. However, in the unmanaged group, non conformity to the treatment for the attack resulted in a cost excess  .Over a ten-year period in United States, hospital inpatient care represented the largest component cost of direct medical expenditures in 1985. However, in 1994, medications were reported to be the largest component cost of direct medical expenditures. The annual estimates increased from approximately $1.4 billion (1985 adjusted dollars) to $2.5 billion. This increase was due to an estimated 103.2% increase in total number of prescribed medications and an estimated 169.3% increase in average unit cost per medication .Our review showed that there are associations between asthma-related costs and the stratification of hospitals by geographic region, ownership, location, and teaching status. In this systematic review, five studies explored the relationship between hospital "characteristics" and the cost of asthma. A large, comprehensive administrative database examined the association of selected hospital characteristics with cost among pediatric patients with asthma and its severity by hospital type in the state of New York. Hospital types were classified into teaching versus non-teaching and private versus public. They reported the mean cost of asthma to be higher in private hospitals,  and non-teaching hospitals . Although after adjustments for patient and hospital covariates, the differences in mean cost between public and private hospitals did not remain significant . In contIn a cross-sectional study, the mean total charges, after adjusting for significant covariates including severity of illness, income, and payer, were found to be significantly higher at investor owned  as opposed to nonprofit  or public hospitals . Furthermore, the average charges were found to be higher at urban teaching  and lower at rural institutions  compared with urban non-teaching hospitals  . HoweverMedications were found to be another major contributor to the cost of asthma. Eighteen studies reported "medications" as forming the largest proportion of the direct costs related to asthma, accounting for 38%–89% of the total cost ,52,56,60In a large-scale study, the costs of asthma medication were found to be the largest cost factor in children  whilst it was reported as being lower in adults. Furthermore, hospital care was found to be a major cost factor in adult patients  . BesidesBesides hospitalization and medications, some patients were shown to have much higher or lower costs, depending on the actual utilization of medical services.Outpatient services provided by hospitals may be in the form of clinics, or be more similar in practice to a group medical practice within a hospital. These outpatient visits provide the same kind of care that is provided in a physician's office; it is thus anticipated that the costs will be the same. In a recent retrospective study, the largest proportion of direct costs was due to outpatient clinic costs (48.5%) and the majority (~76%) of this cost was due to completing pulmonary function tests (44%) and skin-prick tests (34%) . SimilarUnscheduled consultations found to be major components of primary care costs associated with the management of asthma. Prescribed medication for maintenance treatment was also found to contribute significantly to the total asthma related costs. However, the results from studies comparing these two services have given conflicting results. In a large high-quality cohort study in eight countries from the Asia-Pacific region, direct costs of asthma were estimated as: total costs, urgent (unscheduled) versus maintenance costs, and drug versus non-drug costs. The study reported that the urgent care costs were higher than maintenance costs in the participating areas: Singapore, Hong Kong, Malaysia and China, representing 62% of the total costs . In contAnother cohort study classified the encounters into four categories of services: non-urgent outpatient visit, urgent care visit, pharmacy refills, and hospitalization. This study showed that two-thirds of the asthma care costs were attributable to non-urgent outpatient care and prescriptions; only one third were found to be attributable to urgent care and hospitalizations. This was possibly due to better access of enrollees to preventive asthma care, resulting in fewer acute exacerbations requiring emergency department (ED) visits or hospitalizations . SimilarIn this systematic review, we found that adequate insurance coverage in patients with asthma correlated with a reduced use of urgent or emergent care. Two studies investigated the influence of insurance coverage on medical resource utilization and costs. In the first, which was a high-quality cohort study, the patients who were insured reported for every visit that they used more primary care services (at higher costs) and less emergency department services (at lower costs) than patients who were uninsured. However, patients who were uninsured on some visits were reported to have higher primary care, emergency department, and inpatient costs than patients in any other insurance category, suggesting a greater severity of illness among these patients. An additional reason is that they also lacked insurance coverage to purchase more appropriate controller asthma medications . SimilarEconomic assessments of asthma included in the reviewed studies indicated that decreased productivity at work and school represent a considerable proportion of the disease burden, specifically adding to indirect health costs. To accurately assess health-related work impairment, it is important to take into account both time lost from work, or absenteeism, as well as loss of productivity while at work. Productivity losses were measured as the days lost from productive activities because of asthma, the days that patients worked despite asthma symptoms (restricted days), and travel and waiting time associated with receiving outpatient asthma care. Twelve studies included loss of productivity due to absenteeism from work/school as the largest single indirect cost ,48,51,65Children with asthma have much higher indirect costs than the average child as the costs of parents missing work due to the child's asthma is also an indirect cost. In a study conducted in Hungary, high consumption of indirect resources was observed for both the adult and pediatric population. However, the indirect costs represented a much higher proportion of total costs for pediatric patients compared with adult patients (52% vs. 21%). Furthermore, there was a statistically significant increase in the cost of lost work between parents of patients with good control and parents of patients with poor control .In our systematic review, disease of greater severity, was associated with a higher total costs. Twenty-two studies suggested more severe disease to be a major factor influencing the increase in asthma- related costs ,62,66,67The mean annual cost per patient increased as the level of disease control decreased. Seven studies reported that "poor asthma control" was associated with an increase in healthcare and elevated costs ,31,51,54An association between asthma patients with co morbidities and higher costs was also found. Three studies reported that costs increased significantly amongst asthma patients with comorbidities/comorbid conditions ,35,54. IThe high costs among unemployed or retired patients constituted an important finding because these patients were found to be most likely to have a poor overall prognosis in terms of morbidity and mortality. In a study conducted in Switzerland, the highest total costs were observed among jobless patients and those receiving disability payments . SimilarAge was also found to have an association with the cost of asthma, but there were conflicting reports. Younger age was found to be a significant predictor of higher costs in seven studies ,62,70,71Gender was also found to have an association with the cost of asthma. Females were found to have higher direct costs than males, independently of the severity of disease in three high-quality studies ,16,37.We also found a correlation between race and the cost of asthma. Identified Caucasian patients, particularly females in a high-quality study over one year, were found to have significantly more primary care visits (at higher costs) compared with African Americans . In contBesides disease severity, current use of preventive drugs, current use of emergency services and/or current hospitalization was found to be the predictors of costs of childhood asthma . Not surEconomic evaluations of asthma have been performed in many countries. But there are only limited population based studies. The studies reported here are difficult to compare because of differences in study designs, definitions of costs, and different time periods. The present systematic review was aimed at identifying the evidence concerning the economic burden of asthma, as there have been no other systematic reviews in the literature that have reviewed this field and it is a highly emergent aspect of health-services management.Asthma is associated with enormous healthcare expenditures that includes both direct costs, in the form of hospitalizations and medications, and indirect costs, in the form of loss of work which is a combination of directly missed days of work/school that occur during the exacerbation and the loss of future potential earnings associated with both morbidity and mortality.Indirect costs in some studies constitute a major portion of health related costs, and were identified to be higher than direct costs in six studies ,22,51,52In contrast, nine cost of illness studies reported that the direct costs of asthma, associated with hospitalization or medications, to be higher than the indirect costs of asthma ,42,47-50The significant contribution of direct healthcare costs due to hospitalization in studies is not surprising given the inherent high costs associated with acute care versus ambulatory care -57,65,72The fact that the cost of asthma medications is the largest proportion of direct costs of asthma, is likely explained by the fact that there are a relatively large proportion of patients with asthma of whom only a minority are admitted to hospital. In addition the combination of frequent use, the higher cost of newer asthma drugs ,52,56,60The predominance of non urgent office and outpatient costs compared to hospitalization and emergency department charges reported in five reviewed studies might be explained by the better access of patients to preventive asthma care or fewer acute exacerbations requiring hospitalization or emergency care visits ,39,40,43Hospital type and characteristics were reported to have been associated with differences in charges for asthma hospitalizations. Teaching hospitals were found to have higher charges compared with non-teaching hospitals . This isThe higher costs associated with patients admitted to medical centers and regional hospitals than other patients admitted to district hospitals can be explained partly by the tendency of medical centers and regional hospitals to receive a relatively higher proportion of patients suffering from more serious illnesses than district hospitals .In contrast, two studies in the US found no significant differences in costs and charges between teaching and non-teaching hospitals suggesting that, even with the responsibility of providing education, research, and care for indigent patients, teaching hospitals are competitive in their treatment of asthma ,59. One Not surprisingly costs of asthma care were found to increase in the presence of exacerbations and with disease of greater severity ,62,66,67The reasons that children with asthma reported higher healthcare costs compared to non-asthma conditions could be related to: higher use of ambulatory care and medications for upper respiratory tract infections or conditions that can act synergistically with asthma , the use of day-care centers, experiencing more severe asthma on average and higher healthcare use in general, and misclassification of asthma-related encounters as "non-asthma" ,62,70,71Several studies have investigated a variety of potential risk factors associated with a higher risk of direct or indirect costs of asthma including: both old and young ages, female gender, smoking, co-morbid conditions, chronic cough and phlegm, use of peak expiratory flow rate meters, free access to healthcare, low-income status, nonwhite race, asthma symptoms in the past year, longer duration of asthma, controller therapy versus quick therapy, involvement of a pulmonologist in diagnosis or treatment, number of β-agonists and oral corticosteroid prescriptions, and whether inhaled corticosteroids had been prescribed before. The findings that females with asthma spend more on annual asthma care than males might be because of a higher use of acute care facilities due to inadequate medication and poor inhalation skills, or it might be interpreted as females confronted with a chronic illness are more concerned than males and thus, seek medical care more often and have more medicine prescribed.Three more recent studies have been reviewed in this systematic review; however, their findings didn't contribute further to the previous results -75.Despite the availability of effective preventive therapy, hospital admissions from acute asthma are increasing. This might demonstrate that patients with acute asthma exacerbations continue to be treated inappropriately prior to hospital admission.In summary, asthma is not only associated with patient specific impairment, but it is also associated with a significant cost to society. The comparison of studies assessing direct and indirect costs of asthma underscores important facts: hospitalization and medications have been found to be the most important cost driver of direct costs, while work/school absenteeism accounted for the greatest percentage of indirect costs. The cost of asthma was found to be strongly correlated with comorbidities, age, severity of disease, and some other factors. It was also found to vary significantly by hospital ownership, location, and teaching status. A large variation of asthma control can partly be explained by variation in guideline adherence to medication use and deficits of patients' management especially as it relates to access to patient education. Particular interventional strategies such as intensive education of patients and physicians, regular follow-up and preplanned homecare are required to improve quality of life as well as decrease the economic burdens of asthma.ACT: Asthma Control Test; AIDS: Acquired immune deficiency syndrome; AUD: Australian dollar; CAD: Canadian dollar; CDSR: Cochrane Database of Systematic Reviews; CINAHL: Cumulative Index to Nursing and Allied Health Literature; ED: Emergency Department; EUR: The euro currency code; GBP: Great Britain pound; ICER: Incremental cost effectiveness ratio; ICU: Intensive Care Unit; OHE-HEED: Health Economic Evaluation Database; SD: Standard Deviation; UK: United Kingdom; USD: United States dollarThe authors declare that they have no competing interests.KB edited and prepared the final manuscript for publication. KB and KA were responsible for reviewing articles, judging their relevance, assessing their quality, and extracting data. MW assisted in literature search. MF, MW, JS, CM, and LL critically reviewed drafts of this article. MF supervised the literature review, edited the manuscript and provided general oversight. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Anourosorex squamipes) captured in Cao Bang Province, Vietnam, in December 2006, indicated that it is genetically distinct from rodentborne hantaviruses.Sequence analysis of the full-length medium segment and the partial small and large segments of a hantavirus, detected by reverse transcription–PCR in lung tissues of the Chinese mole shrew ( Suncus murinus) (Sorex araneus), alpine shrew , Eurasian water shrew (Neomys fodiens), and common mole  (Crocidura theresae) in Guinea (Blarina brevicauda) in the United States (C. lasiura), captured along the Imjin River near the demilitarized zone in South Korea .Insectivores (or soricomorphs) have been largely ignored as being important in the evolutionary dynamics of hantaviruses, despite the isolation of Thottapalayam virus (TPMV) from the Asian house shrew (Crocidura spp.), 3 Chinese mole shrews (Anourosorex squamipes), and 12 long-nosed moles (Euroscaptor longirostris), captured in northern, central, and southern Vietnam during November and December 2006. RNA, extracted from 20–50 mg of each tissue by using the RNA-Bee isolation kit , was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase  and the primer 5′-TAGTAGTAGACTCC-3′. Oligonucleotide primers for subsequent nested PCR were designed from consensus regions of TPMV and other hantaviruses , from 24 soricomorphs, including 9 white-toothed shrews , 1 μg of cDNA, and 10 pmol of each primer. Initial denaturation, at 94°C for 5 min, was followed by touchdown cycling with denaturation at 94°C for 40 s, annealing from 50°C to 37°C for 40 s, elongation at 68°C for 1 min 20 s, then 25 cycles of denaturation at 94°C for 40 s, annealing at 40°C for 40 s, and elongation at 68°C for 1 min 20 s in a Mastercycler ep gradient S . PCR products were purified by the Wizard PCR Preps DNA Purification System (Promega). DNA sequencing of at least 3 clones of each amplicon was performed in both directions with the dye primer cycle sequencing ready reaction kit  on an automated sequencer   medium (M) segment was amplified from lung tissues of 3 Chinese mole shrews, captured in Thanh Cong commune, Nguyen Binh District, Cao Bang Province, along the southern border of the People’s Republic of China. Designated Cao Bang virus (CBNV), the newly identified hantavirus exhibited low nucleotide and amino acid sequence similarity to representative hantaviruses harbored by Murinae, Arvicolinae, Neotominae, and Sigmodontinae rodents, ranging from 62.6% (nt) and 61.2% (aa) for Hantaan virus (HTNV) 76–118; 62.3% (nt) and 61.7% (aa) for Dobrava virus (DOBV) Greece to 58.1% (nt) and 52.0% (aa) for Puumala virus Sotkamo; and 58.8% (nt) and 54.7% (aa) for Sin Nombre virus NMH10 .Pairwise alignment and comparison of a 1,185-nt coding region of the small (S) segment showed similar degrees of sequence identity between CBNV and rodentborne hantaviruses, ranging from 61.4% for HTNV 76–118 to 58.0% for TULV (Tula virus) M5302v. Much higher sequence similarity was found in a 412-nt coding region of the large (L) segment, ranging from 72.6% for HTNV 76–118 and 75.2% for DOBV Greece, to 67.2%–70.1% and 67.7%–71.6% for hantaviruses harbored by Arvicolinae, Neotominae, and Sigmodontinae rodents, respectively. CBNV sequences were similarly divergent from Tanganya virus (TGNV) Tan826: for S segment, 63.5% (nt) and 65.0% (aa) similarity; for L segment, 71.3% (nt) and 77.0% (aa) similarity.Phylogenetic trees based on sequences of the full-length M segment and partial S and L segments, generated by the maximum likelihood and neighbor-joining methods using the GTR+I+G model of evolution, showed similar topologies supported by bootstrap analysis, in which CBNV was relatively distinct from rodentborne and other shrewborne hantaviruses . A stronDesigning suitable primers for the amplification of CBNV presented unanticipated challenges. Ironically, the recently acquired full genome of TPMV  was not particularly helpful, since CBNV was genetically more divergent from TPMV than from well-characterized rodentborne hantaviruses . Also, because the tissues were collected in RNAlater, virus isolation attempts could not be performed. As such, progress in obtaining the full-length sequence of CBNV has been slow.A forest-dwelling soricine typically residing at elevations of 1,500–3,000 m, the Chinese mole shrew  has a vast geographic range, extending from western and central People’s Republic of China, northern Myanmar, northern Thailand, Assam, Bhutan, northern Vietnam, Taiwan, and possibly Lao People’s Democratic Republic. That a hantavirus has been identified in the Chinese mole shrew was not completely unexpected, in view of the isolation of a HTNV-like virus from this species in Sichuan Province in 1986 (B. brevicauda), Eliot’s short-tailed shrew (B. hylophaga), masked shrew (Sorex cinereus), and montane shrew (S. monticolus) in the United States —as well as in the Eurasian common shrew in Switzerland, Hungary, and Finland , the Ussuri shrew in South Korea , the Asian house shrew in India (Hantavirus genus will provide additional insights into the phylogeography and co-evolution of hantaviruses and their soricid reservoir hosts. One or more of these newfound shrewborne viruses may yield valuable clues about the molecular determinants of hantavirus pathogenesis.Viewed within the context of newly identified, genetically distinct hantaviruses in the northern short-tailed shrew (


Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier® ELISA Cp. abortus serum verification kit.Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Cp. abortus were detected.No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.The results suggest that  Bovine chlamydiosis has been associated with several disease manifestations . ReproduChlamydophila (Cp.) abortus and Cp. pecorum are known to infect cattle and are suggested to be ubiquitous [Cp. psittaci infections in cattle have been reported [Cp. abortus is the cause of Ovine Enzootic Abortion (OEA), the major infectious cause of abortion and lamb loss with great economic losses in many sheep-producing countries [Cp. pecorum has foremost been associated with polyarthritis, encephalitis and inapparent intestinal infection, and the impact by Cp. psittaci in ruminants is yet to be investigated.The two species iquitous ,19. Morereported ,21. All reported ,23. Cp. ountries . Cp. pecChlamydophila spp. by venereal route. Additionally, artificial insemination (AI) is performed yearly on more than 95% of the approximately 400,000 Swedish dairy cows [Chlamydophila spp. via this route, it is important to determine whether breeding bulls are infected through screening of semen before AI in order to minimize this risk. The aim of this study was to investigate the presence of chlamydial agent in semen and in tissue of cauda epididymidis and to estimate the seroprevalence of Cp. abortus in Swedish bulls.Each year about 80 top-ranked performance-tested yearling beef bulls are sold all over Sweden, mainly to pedigree breeders, after six months of testing at the only performance testing station in the country. These performance-tested bulls represent the best-documented beef bulls with the highest impact on the breeding programme in Sweden and are therefore important potential transmitters of iry cows . As therThis study comprises samples from a subset of 166 beef bulls from 124 herds from different parts of Sweden that were taken to the only performance testing station in Sweden in September 2002. On arrival the bulls were approximately six months old. They were divided into groups, based on breed and body weight and placed in ten adjacent semi-outdoor pens under the same roof. The bulls were weighed every second week throughout the testing period (September-March) and at the end of the period, an individual growth index was calculated. Bulls with fast growth rates were sold at livestock auction and bulls with growth indexes below the threshold, stated by the breeders' organisations, were either slaughtered or returned to their owners. In total, 43 of the beef bulls that were sent to slaughter were included in this study . The daily growth rates of these bulls were somewhat lower than the bulls sold at auction, but were still higher than the growth rates of non-tested beef sires in Sweden . BecauseTestes and epididymides were removed at the time of slaughter and immediately put in a container with crushed ice and transported refrigerated to the laboratory where they arrived the next day. An incision (approx. 1.5 cm long and 0.5 cm deep) was made with a scalpel blade in the middle, distal part of the cauda epididymides , and a shttp://www.vikinggenetics.com. Semen samples were stored at -70°C prior to preparation for analysis by real-time polymerase chain reaction (PCR). Sera for serology were stored at -20°C until analysed.Semen samples (0.2 ml payettes) and sera from 21 dairy bulls about 1 year old (Swedish Holstein and Swedish Red) in service, were submitted to the laboratory from one of the only two semen producing companies in Sweden, Svensk Avel Chlamydiaceae-specific real-time PCR protocol developed by Everett and others [DNA was extracted from semen and cauda epididymidis samples for PCR analysis using a High Pure Template Preparation kit, following manufacturer's instructions  and stored at -20°C. Analyses were performed using a d others , targetiAn internal amplification control (mimic) was constructed and used to detect false negative PCR results, as previously described . The pri2, 0.2 mM of each of the four dNTP, 0.15 μM of each of the primer TQF and TQR, 0.25 μl (1.25 U) of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 0.1 μM of each probe. Reaction mixtures were placed in a Rotor-Gene 3000  and amplification was performed according to the protocol of Everett and others [The 25-μl PCR mixture comprised 2.5 μl of 10× PCR-buffer II , 2.5 mM MgCld others . The resCp. abortus .The sensitivity of the PCR was estimated to one inclusion forming unit (IFU) per PCR by spiking semen and tissue samples prior to DNA extraction with ten-fold dilutions of ® ELISA Chlamydophila abortus serum verification kit  was applied. The ELISA uses a recombinant fragment of an 80-90 kDa polymorphic outer membrane protein and detects antibodies against Cp. abortus. The assay was used according to the manufacturer's instruction with S/P% values ≥ than 100 as positive for cattle.For detection of antibodies the PourquierAll 21 semen and 43 cauda epididymidis samples were negative in the PCR. The internal amplification control (mimic) worked well for all samples analysed.None of the 21 and 43 paired-sera from dairy and beef bulls, respectively, were positive in the antibody detection assay. Most samples were clustered well below the cut-off value 100. Only six samples had S/P% above 20, where 42 was the highest value.Cp. abortus nor Cp. pecorum was detected in 273 semen samples from bulls at five AI centres. On the other hand, the results contradict those reported from other investigations performed in apparently healthy bulls. In Lithuania as much as 29.8% of 47 tested bulls had chlamydial agent in their semen, as judged by PCR [In this study we found no presence of chlamydial agent in any semen or cauda epididymidis tissue samples, i.e. all samples were negative by real-time PCR. This is in concordance with an Austrian study  where ned by PCR , and chld by PCR . In Germd by PCR ,23.The sensitivity of the PCR assay was estimated to 1 IFU per PCR with no indication of potential inhibitory factors. In a previous investigation of cows from dairy herds with reproductive disorders we identified positive specimens, including vaginal swabs, placenta and milk when using the same PCR assay . MoreoveCp. abortus ELISA assay with values far below the cut-off value. The specificity of the test has been reported to be 100% when used to analyse Scottish sheep documented free of Cp. abortus [Cp. abortus, were analysed [Cp. abortus infected sheep [Cp. abortus is not present in Swedish beef cattle herds. Moreover, the absence of seropositives among the analysed dairy bulls indicates that Cp. abortus is very rare, or absent, in Swedish bulls. These results are in agreement with a previous study in Swedish dairy cows where only 2 out of 525 sera were positive in the same ELISA and only Cp. pecorum were confirmed in vaginal swabs [All sera were negative in the  abortus  and 90% analysed . The sened sheep ,36., andal swabs .Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.This study suggest the risk for venereal transmission of chlamydial infection through Swedish bull semen is low. However, because The authors declare that they have no competing interests.ACK participated in the design and coordination of the study, drafted and rewrote the manuscript, carried out the PCR and interpreted the results. SE implemented the PCR systems, constructed the mimic and interpreted the results. CB and SA participated in the design and coordination of the study. YP sampled and wrote about the beef bulls. All authors have been involved in revising the manuscript.


This organism should be recognized as an emerging pathogen in the United States. Cryptococcus gattii has emerged as a human and animal pathogen in the Pacific Northwest. First recognized on Vancouver Island, British Columbia, Canada, it now involves mainland British Columbia, and Washington and Oregon in the United States. In Canada, the incidence of disease has been one of the highest worldwide. In the United States, lack of cryptococcal species identification and case surveillance limit our knowledge of C. gattii epidemiology. Infections in the Pacific Northwest are caused by multiple genotypes, but the major strain is genetically novel and may have emerged recently in association with unique mating or environmental changes. C. gattii disease affects immunocompromised and immunocompetent persons, causing substantial illness and death. Successful management requires an aggressive medical and surgical approach and consideration of potentially variable antifungal drug susceptibilities. We summarize the study results of a group of investigators and review current knowledge with the goal of increasing awareness and highlighting areas where further knowledge is required. Cryptococcus gattii (formerly C. neoformans var. gattii) , A (C. neoformans var. grubii), D (C. neoformans var. neoformans), and the hybrid diploid AD  more commonly than C. neoformans, and C. gattii disease is more often associated with neurologic sequelae, frequently requiring aggressive neurosurgical management , parts of Latin America , southern California, Mexico, Hawaii, Central and South Africa, and certain parts of Europe   medium .Our understanding of the epidemiology of cryptococcosis is limited by our currently used diagnostic methods. Diagnosis of cryptococcosis frequently relies on direct microscopy, culture of clinical samples, or detection of cryptococcal antigen in body fluids. However, the organism is not identified to species level in many clinical microbiology laboratories. One biochemical characteristic that distinguishes C. gattii epidemiology, have used the techniques of PCR fingerprinting  and VGIIb (≈10%), representing 2 independent clonal populations  , and water . Another similar isolate was found in the environment in San Francisco, California, in 1990 (strain CBS7750) (C. gattii was present in the environment (and may have been a cause of human disease) in this part of the United States for many years before recognition of the Vancouver Island emergence  was diagnosed in a resident of Orcas Island, Washington  and MLST .In early 2006, the first US case of human infection by a C. gattii disease have been identified in Oregon since 2004. During 2004–2005, two cases of culture-confirmed C. gattii infection were recorded in the United States. These case-patients did not report exposure to Vancouver Island or other C. gattii–endemic areas. Isolates from these case-patients (both Oregon residents) were VGII, but they were genetically distinct from Vancouver Island VGII isolates and their relationship to the outbreak is unknown  . In Oregon, VGIIa C. gattii infection was found in 2007 in 1 cat in Lebanon, 1 dog in Salem, and 2 alpacas in McMinnville and Sherwood  and above freezing daily winter average temperatures . Similar climates with comparable temperature and rainfall extend further south into Washington and Oregon. Additionally, the San Juan Islands, Puget Sound, and the Willamette Valley contain plant diversity ecologically similar to that on Vancouver Island and in mainland British Columbia, lending support to the idea that C. gattii may have niche areas suitable for colonization in the broader Pacific Northwest   of 2,033 non-Vancouver Island environmental samples  were positive for Cryptococcus species are haploid yeasts that predominantly reproduce asexually (through mitosis and budding). However, they also possess a bipolar mating system, with mating types a or α. These mating types are capable of completing a sexual (meiotic) cycle that is either heterosexual (between a and α) or unisexual  . However, to gather sufficient information about the true incidence of this disease and to perform necessary clinical studies, a coordinated, systematic, regional approach to human and veterinary surveillance is needed.Although formal C. gattii isolates isolated from environmental, animal, and human sources. Additional central repositories of C. gattii strains and clinical databases would be useful for investigators in the field.Currently, the University of British Columbia and the British Columbia Centre for Disease Control hold a large collection of C gattii disease are necessary for studying the pathogenicity of genetically diverse isolates and host immunity, as well as to answer more fundamental questions about the disease. Laboratory-based efforts should also focus on development of better diagnostic aids, such as a reliable system to isolate DNA from fixed tissue. Development of species-specific serologic tests would enable seroprevalence studies for a better understanding of epidemiology and disease mechanisms. Studies of soil persistence and propagation of the organism and those that address the nature and factors controlling the infectious propagule would be informative  lineages might hold the key to understanding the differences in niches, virulence, mating, and the ability to cause an outbreak.C. gattii disease was introduced and spread within the Pacific Northwest, including the possible role of global climate change, deforestation and increased land use, as well as factors related to the biology of the organism and changing host susceptibility. This knowledge, acquired through environmental and genomic research studies, would help identify high-risk groups or regions, and provide information on risk-reduction and improvement of diagnosis and treatment.From a long-term public health perspective, it is important to gain an understanding of how C. gattii has become endemic to the Pacific Northwest. Efforts to understand the emergence on Vancouver Island and mainland British Columbia have generated valuable information. However, many questions remain unanswered. The Centers for Disease Control and Prevention  is currently working in conjunction with state public health departments to enable clinical (human and veterinary) surveillance. Additional organized national and international efforts are needed. As the first step, C. gattii should be recognized as an emerging infection in the United States and declared a priority pathogen in the eyes of funding agencies.


Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.The yellow fever virus, a member of the genus YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow  The yellow fever 17D virus is attenuated and used for human vaccination for 70 years. Some of the outstanding properties of this vaccine include limited viral replication in the host but with significant expansion and dissemination of the viral mass yielding a robust and long-lived immune response -5. The vfg loop of the E protein ]. TM1 would serve as an anchor to E whereas TM2 would act as a signal sequence for NS1, and interactions between the two have a role in viral envelope formation . The segractions . Length ractions . TherefoHerein we describe the design, construction and regeneration of live YF 17D and 17D-Dengue 4 (YF17D/DEN4) viruses bearing the green fluorescent protein gene between E and NS1. We have characterized foreign gene expression and genetic stability as well as recombinant virus immunogenicity.For the flaviviruses, the polyprotein precursor transverses the ER membrane at various points being proteolytically processed in the ER lumen by cellular signal peptidases and in the cytoplasmic side by viral NS2B/NS3 protease. Protein secretion and processing require the presence of functional motifs. The design of a foreign sequence insertion in the YF 17D virus E and NS1 intergenic region considered the presence of such motifs as well as amino acid sequence conservation flanking this location. Figure Figure Our approach towards the regeneration of viable virus with a gene insertion between E and NS1 was to duplicate the first 9 amino acids of NS1 at the amino-terminus of the EGFP gene and the whole E protein stem-anchor domain at its carboxi-terminus Fig. . This stIn vitro transcribed RNA was used to transfect cultured Vero cells. When the cytophatic effect (CPE) was widespread, the viability of the constructs could be visualized by fluorescence microscopy of the Vero cell monolayers. In the case of the YF17D/Esa/5.1glic virus it was performed at 72 h post-infection. This viral stock, called P1, was used for a second passage in Vero cells, or P2, which resulted in a viral stock with the titer of 6.18 log10 PFU/mLThe growth capacity of the recombinant YF17D/Esa/5.1glic virus was assessed comparatively to two other viruses, YF 17DD vaccine and YF17D/E200T3 . Three iP = 0.095; P = 0.576 and P = 0.3890, respectively). But at 48 h, 72 h and 96 h the differences in virus yields were statistically significantly . The recombinant YF17D/Esa/5.1glic virus displayed a small plaque phenotype (0.99 ± 0.2 mm) when compared to the intermediate size of YF17D/E200T3 (1.65 ± 0.3 mm) and the large plaques of the YF 17DD virus (2.80 ± 0.7 mm).At 24 h, 120 h and 144 h time points there were no significant difference between the viral titers of YF 17DD vaccine virus and YF17D/Esa/5.1glic . It is noteworthy that the immunization with YF 17D/Esa/5.1glic virus elicited antibodies against EGFP in 80 % of the animals with titers varying from 26 to 3,535 ng/mL . These results suggest the continuous presence of the EGFP gene in the recombinant virus genome and its expression throughout the passages. However, as we continued with these two independent serial passage lines in Vero cells up to the fifteenth one, it was possible to demonstrate a change in the total EGFP+ YF+ labeled cells, which varied from 83 % to 84 % at the tenth passage to 1 % and 20 %, at the fifteenth, respectively (data not shown).We have studied the genetic stability of YF17D/Esa 5.1glic virus by two independent serial passages of this virus in Vero cells up to the tenth passage Fig. . We usedTo better characterize the genetic stability of the YF17D/Esa/5.1glic virus, we set up a serial passage experiment in Vero cells with 5 plaque purified viral clones. All Vero cell cultures infected with each of the 5 cloned viruses exhibited double EGFP and viral antigen fluorescence. The double fluorescence ratio varied from 95 to 99% in cells infected with cloned viruses at their fifth passage. But, at the tenth passage, two cloned viruses have exhibited a double labeling percentage of 7 % and 33 %, suggesting the continuous loss of the foreign sequence in this interval (data not shown). However, the other three cloned virus samples displayed 77 %, 93 % and 80 % of double gated cells at the tenth passage (data not shown), indicating again genetic stability of the EGFP-bearing recombinant virus population.in vitro transcription and transfection of Vero cells with RNA. The chimeric YF17D/DEN 4/Esa/6 construct could only be recovered after trypsinization of the RNA-transfected cell monolayer with an additional incubation of 96 h when CPE became evident. This viral stock, called P1, was used for a second passage in Vero cells, or P2, with a titer of 6.48 log10 PFU/mL. Passage 2 virus was used for further analysis.To verify whether this strategy might be applicable to clone foreign sequences in other flavivirus genomes, we have constructed a recombinant YF17D/DEN4/Esa/EGFP virus, in which the YF prM/E genes were replaced by the homologous genes of the DEN type 4 virus with the EGFP cassete being inserted in the same E/NS1 intergenic region Fig. . It is n10 PFU/mL, respectively, while the recombinant YF17D/DEN4/Esa/6 virus, at 96 hours after infection displayed a viral titer of 6.3 ± 0.1 log10 PFU/mL .Aiming at the characterization of the growth capability of the YF/DEN4/Esa/6 virus in comparison to the YF 17DD vaccine virus and parental chimeric YF17D/DEN4 virus Vero cell monolayers were infected with these viruses at MOI of 0.02. The YF 17DD and 17D/DEN4 viruses peaked at 72 hours after infection, with titers of 7.2 ± 0.3 and 6.7 ± 0.4 logL Figure . At all The genetic stability of the chimeric YF17D/DEN4/Esa/6 virus was assessed by two series of independent passages in Vero cells up to the twentieth passage. The expected length of DNA amplicon containing the EGFP expression cassete is 2,046 bp, while the same region in the parental YF17D/DEN4 virus is 1,017 bp long. As can be observed in Figure The yellow fever virus has been considered as an appealing viral vector for the development of new human vaccines . The mosDuring viral RNA translation, the flavivirus polyprotein precursor transverses the ER membrane at various points being proteolytically processed in the ER lumen by cellular signalases and at the cytoplasmic side by the viral NS2B/NS3 protease . The E pth passage (YF17D/DEN4/Esa/6). The flow cytometry data for cells infected with YF17D/Esa/5.1glic supports the genetic stability of the insert up to the tenth passage. It is possible to produce seed lots intended for industrial production starting from cDNA with 4 passages methionine  for one hour. The cells were washed once and incubated with 10 mL Earle's 199 medium with 5% FCS for 3 h at 37°C in a CO2 incubator. The supernatants were removed, centrifuged 5 min at 300 g at 4°C and protease inhibitors were added . The adherent cells were scraped off and lysed under nondenaturing conditions in the presence of the same protease inhibitors.Vero cells were infected at a multiplicity of 0.5 PFU/cell in 60 mm dishes  and a rabbit polyclonal antiserum directed against GFP (Clontech). Immunoprecipitates were fractionated with protein A-agarose (Invitrogen) and analyzed by 10% SDS-PAGE. Gels were treated with sodium salicylate for fluorographic detection.50) [P < 0.05.Groups of ten four-week old BALB/c mice  were subcutaneously injected with two doses of 100,000 PFU in 100 μL of YF17D/Esa/5.1glic or YF 17DD viruses with an interval of 15 days. Two weeks after the last immunization, mice were bled from the retrorbital vein, serum samples were treated for 30 minutes at 56°C and stored at -20°C. YF neutralizing antibody titer was determined by plaque reduction neutralization test (PRNT50) . The valAequoria victoria (Clontech) diluted in 100 μL carbonate buffer 0.05 M pH 9.6. After overnight incubation at room temperature, the plates were washed three times with phosphate-buffered saline containing 0.05% (v/v) of Tween-20 (PBS-Tween), and blocked at 37°C for two hours with PBS containing 5% (w/v) non-fat milk with 1% (w/v) of bovine serum albumin . The assays were performed in duplicate and every plate was composed of the respective pooled sera of each experimental group. The positive and negative controls consisted respectively of the mouse monoclonal IgG2a antibody (JL-8) specific for GFP and pooled sera from animals immunized with culture medium (Clontech). Pooled sera were analyzed in serial dilutions from 1:20 to 1:2,560, and the JL-8 monoclonal antibody was employed in the range from 10 to 0.78 ng. After a two-hour incubation at room temperature, unbound antibodies were washed away with PBS-Tween, and diluted 1:500 peroxidase-conjugated goat anti-mouse IgG heavy and light chain (Kirkegaard and Perry), was added to each well. After one-hour incubation at room temperature, the excess labeled antibody was removed by washing, and the reaction was developed with o-phenylenediamine (Sigma Co.) and 1 μL/ml H2O2 (Merck). After 15 min, 2 M H2SO4 solution was added to stop the reaction and the plates were read at 492 nm on VERSAmax ELISA reader (Molecular Devices). Every ELISA plate contained a positive column of serially diluted JL-8 monoclonal antibody, which provided the standard curve. The different standard curves were analyzed by linear regression to check the linearity of the data and then used to determine the titers in the experimental groups. Therefore, the EGFP antibody titers were expressed in ng/mL based upon the curve established for the JL-8 monoclonal antibody specific to EGFP.Antibodies to the EGFP heterologous protein were detected by ELISA using microtiter plates (Costar) coated with 10 ng/well of the recombinant GFP of All animal studies were carried out according to a protocol reviewed and approved by the Institutional Committee for Experimentation and Care of Research Animals (CEUA-FIOCRUZ: P0112/02).The author(s) have declared that the present methodology is the subject of a patent application having as authors MCB and RG and the Oswaldo Cruz Foundation as the sponsoring institution.MCB designed the viral constructions and the study, coordinated the study and drafted the manuscript; SMM carried out the YF virus genome cloning work and the flow cytometry analysis; GFT performed the confocal microscopy analysis and the EGFP-ELISA studies; AAR was engaged in the YF/DEN4 virus construction and related studies; ASD was responsible for nucleotide sequencing, assisted in animal studies and helped with the ELISA analysis; PJO performed neutralization plaque assays and data analysis; MSF discussed and assisted in animal studies; CFK designed flow cytometry analysis and led data interpretation; RG designed the viral constructions and helped to coordinate the study and manuscript draft. All authors read and approved the final manuscript.


Picornaviridae contains well-known human pathogens . In addition, this family contains a number of viruses that infect animals, including members of the genus Cardiovirus such as Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelits virus (TMEV). The latter are important murine pathogens that cause myocarditis, type 1 diabetes and chronic inflammation in the brains, mimicking multiple sclerosis. Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). The virus is genetically related to Theiler's virus and classified as a new species in the genus Cardiovirus, which until the discovery of SAFV did not contain human viruses. By analogy with the rodent cardioviruses, SAFV may be a relevant new human pathogen. Thus far, SAFVs have sporadically been detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance remained unclear. Here we describe the first cultivated SAFV type 3 (SAFV-3) isolate, its growth characteristics, full-length sequence, and epidemiology. Unlike the previously isolated SAFV-1 and -2 viruses, SAFV-3 showed efficient growth in several cell lines with a clear cytopathic effect. The latter allowed us to conduct a large-scale serological survey by a virus-neutralization assay. This survey showed that infection by SAFV-3 occurs early in life (>75% positive at 24 months) and that the seroprevalence reaches >90% in older children and adults. Neutralizing antibodies were found in serum samples collected in several countries in Europe, Africa, and Asia. In conclusion, this study describes the first cultivated SAFV-3 isolate, its full-length sequence, and epidemiology. SAFV-3 is a highly common and widespread human virus causing infection in early childhood. This finding has important implications for understanding the impact of these ubiquitous viruses and their possible role in acute and/or chronic disease.The family  Cardiovirus genus, which until this recent discovery did not contain human viruses. Theiler's virus is an important mouse pathogen that causes chronic inflammation in the brains, closely resembling multiple sclerosis in humans. By analogy, SAFV may be a relevant human pathogen. Thus far, SAFVs have been sporadically detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance have remained unclear. Here we describe the first SAFV type 3 (SAFV-3) isolate, its growth characteristics in cell lines, full-length RNA-sequence, and epidemiology. Unlike the previously isolated SAFV-1 and SAFV-2, SAFV-3 grows well in cell lines, resulting in cell damage. This feature enabled us to conduct a large-scale serological survey for virus-neutralizing antibodies. This survey showed that SAFV-3 infection occurs early in life and that >90% of children >2 years and adults had antibodies. Neutralizing antibodies were found in serum samples collected in several countries in three continents. Hence, we concluded that SAFV-3 is a genuine and widespread human virus causing infection early in life.Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). Picornaviruses are small RNA viruses with poliovirus as prototype. Saffold virus is genetically related to Theiler's virus, a member of the  Insight into the potential role of these so-called “orphan” viruses in disease requires a detailed understanding of their genetic diversity and epidemiology. Classically, the association of an infectious agent with disease had to fulfill Koch's postulates, a concept that is no longer tenable in modern times. The clinical outcome of a virus infection may depend upon the conditions under which the infection is acquired: For example, poliomyelitis was seldom observed under conditions of poor sanitation, congenital rubella syndrome is a consequence of postponed childhood infection and some types of cancer are late events in which certain viruses play a crucial role. Moreover, it requires detailed insight in viral diversity, since it is well known that minor differences in the genetic make-up of viruses can cause major differences in their pathogenicity. The latter holds especially for RNA viruses such as the picornaviruses which due to their high mutation and recombination rates show remarkable genetic plasticity which may lead to serious pathology merely by accident Recent advances in molecular detection methods , Parechovirus, Hepatovirus, and Kobuvirus. This family also contains economically important animal pathogens such as Foot-and-mouth-disease virus, a species belonging to the Aphthovirus genus. Other well-known animal pathogens are Theilovirus and Encephalomyocarditis virus (EMCV), two species that belong to the Cardiovirus genus and are associated with disease in rodents and swine. The Theilovirus species is represented by Theiler's murine encephalomyelitis virus (TMEV) and rat encephalomyelitis virus . TMEV is an enteric pathogen that primarily causes asymptomatic infections of the alimentary tract. However, extra-intestinal infection can occur and produce acute fatal encephalomyelitis or a chronic demyelinating disease relevant for multiple sclerosis, depending on the TMEV strain involved The family The question of whether authentic human cardioviruses exist has remained unclear for a long time. A Theiler's-like cardiovirus, named Vilyuisk human encephalomyelitis virus (VHEV), has been implicated in an outbreak of a neurodegenerative disease among the Yakuts people in Vilyuisk, Siberia, in the 1950s. However, this virus, which was isolated upon multiple passages in mice and cell cultures, shows close relationship with TRV and TMEV, raising the possibility that it represents a contaminating animal cardiovirus Here we report the first isolation, full-length sequence, characterization, and epidemiology of a SAFV-3 isolate. Evidence is provide that this SAFV is a ubiquitous human virus causing infection early in life. The implications of this finding for understanding its biology and possible associations with disease are discussed.In December 2007, a virus was isolated from a stool sample obtained from a 13 month-old boy who presented with vomiting. The sample was routinely sent in to exclude viral gastro-enteritis but the complaints were not due to an infection but to an anatomical mal-position of the jejunum. Culture on human embryonic lung fibroblasts (HELF) and tertiary monkey kidney cells (tMK) yielded a virus that was acid-stable, chloroform resistant and that by electron microscopy showed the size (approximately 30 nm) typical of a picornavirus (data not shown). However, the virus isolate could not be typed as an enterovirus or parechovirus.To identify and characterize the unknown virus, which was provisionally named NL2007, the tMK isolate was propagated on HeLa cells and the virus was partially purified by centrifugation through a sucrose cushion. Nucleic acids were isolated from the pellet, reverse transcribed and amplified using a random PCR method. PCR fragments were cloned and sequenced. Based on a sequence comparison (BLASTn search), the virus was identified as a SAFV A full genomic sequence was obtained by a genome walking method on amplified, partially overlapping fragments of 1 to 1.5 kb. The sequence of the end of the 5′ UTR was determined using a 5′-RACE amplification protocol. The genome contained a 5′ UTR of 1,042 nucleotides (nt), an open reading frame (ORF) of 6,888 nt, and a 3′ UTR of 121 nt followed by a poly(A) tail (GenBank accession number FM207487).Alignments and phylogenetic analyses were performed with available SAFV sequences  is similar to that of the other cardioviruses cis-acting replication element (CRE), involved in uridylylation of the peptide primer, VPg The 5′ and 3′ UTRs of SAFV-3(NL-2007) are highly conserved to those of SAFV-1 and -2 . The 5′ UTR contains a type II IRES typical for the cardioviruses. Secondary structures of the RNA replication elements in the UTRs, as predicted by MFOLD The leader (L) protein, an interferon antagonist The overall nt identity in the P2 and P3 regions to SAFV-1 and -2 is 82–90%. At the aa level, the P2 and P3 region proteins in the SAFVs are even 95–100% identical. The corresponding regions of TMEV, TRV, and VHEV were more divergent (61–90% identity at aa level) , although the P2 and P3 region proteins of SAFVs were more closely related to those of TRV than to those of TMEV and VHEV. In contrast to the high similarity for P2 and P3, SAFV-3(NL2007) differs significantly from SAFV-1 and SAFV-2 in the P1 region. The divergence is highest in VP1  and VP2  and lowest in VP4 region , which is located at the interior of the capsid. The identity of SAFV-3(NL2007) with the Germany SAFV-3 strains (2223 and 2273) is 91% (nt) and 98% (aa), whereas that with the US SAFV-3 strains (UC1 and UC5) is 86% (nt) and 97% (aa).Cardioviruses contain 4 small loops at their outer surface that are important sites for recognition by neutralizing antibodies Thus far, SAFVs have been shown difficult to propagate on cell lines. Only two SAFVs have been propagated in cell culture, a SAFV-1 in human fetal diploid kidney (HFDK) cells p<0.0005, unpaired t-test, two-tailed). Of 90 children between 4–10 years of age, 92% had neutralizing antibodies against SAFV-3 and 98% of 60 adults (p<0.05) (Growth properties of SAFV-3(NL2007) with a clear CPE in HeLa cells made it possible to conduct a serological survey by virus neutralization (VN). A total of 210 serum samples sent to our laboratory for routine diagnostics, were tested in the VN assay. These included serum samples from 60 adults  collected in 2004 and serum samples from 120 paediatric patients, of different age groups: 2, 4, 8 and 10 year of age, 30 samples each, collected in 2006–2007. In addition, serum samples obtained in 2006–2007 from 30 infants, 9 months of age, were tested. At this age, serum IgG levels start to rise from a physiological nadir and remaining maternal antibody levels are low. As is shown in 0 adults Table 1.Next, we investigated the prevalence of SAFV-3 antibodies in other countries. Serum samples from 30 children from Finland (age 24–30 months), collected in 1997–1998, were tested. Furthermore, we tested serum samples from 72 African individuals  and from 63 Indonesian individuals . Neutralizing antibodies were found in 77% of Finnish children and 100% of people living in Africa or Indonesia Table 1,We also investigated the possible occurrence of SAFV-3 antibodies in blood specimens from laboratory rodents. For this purpose, we used pooled serum samples from rats, mice, hamsters, guinea pigs, and rabbits. No antibodies were observed in the samples from either of these rodent species. In addition, we tested pooled serum samples from rats and mice containing high titer antibodies to TMEV or EMCV. Again, no SAFV-3 neutralizing antibodies were detected (data not shown). Together, these data suggest that it is unlikely that SAFV-3 is a common virus in rodents. Moreover, these data further emphasize the specificity of our neutralization assay.The family Picornaviridae contains a large number of notorious human and animal pathogens. The recent identification of human cardioviruses, Saffold viruses, has prompted studies to establish an association between these viruses and disease. Current information on SAFVs is almost exclusively based on molecular detection of these viruses in respiratory and stool samples, where virus is found at low frequency and mainly in children under 6 years of age The serological survey was performed with a SAFV-3 isolate that we isolated and characterized. Our SAFV-3 isolate is the first reported SAFV that efficiently replicates in cell lines and produces a clear cytopathic effect, thereby allowing to conduct a large-scale survey by means of a specific virus-neutralization assay. Using the assay, we showed that SAFV-3 is a highly common and wide-spread human virus causing infection in early childhood: the seroprevalence of SAFV-3 reaches high levels (>90%) in young children and adults. Nine months after birth, maternal antibodies have almost disappeared. In accordance, at nine months of age, many infants that we tested were seronegative and those that had antibody showed low titers which may still have been of maternal origin. A steep rise was found in 2-year-old children, both in seroprevalence and in titers, which indicates that infection occurs frequently and early in life. It is too early to state that SAFV infections occur before the age of 2 years because age of infection can be influenced by periodicity of outbreaks with a particular genotype (e.g. SAFV-3). The latter could explain why the Finnish children in 1997/98 showed a somewhat lower seroprevalence. Alternatively, the low population density in Finland compared to the Netherlands may have limited the speed of circulation of the virus. High rates of SAFV-3 antibody were furthermore found in African countries and in Indonesia, strongly supporting that SAFVs are ubiquitous human viruses with a global prevalence: SAFVs have now been reported from countries in North- and South-America, Europe, South-Asia and Africa A high seroprevalence contrasts with the low numbers (<1%) of respiratory and fecal samples that tested positive by PCR, at least in developed countries Another aspect of consideration is the specificity of serology. Virus neutralization tests are highly specific and can discriminate serotypes within a single species Our serological data are consistent with infection early in life which is corroborated by PCR data being mainly positive in children below 6 years of age human enterovirus C (HEV-C) species which includes coxsackie A viruses causing mild respiratory symptoms, as well as the polioviruses which can cause severe neurological disease and paralysis Although enteroviruses and parechoviruses mostly cause asymptomatic infections, they can cause severe pathology depending on the conditions of infection, which involve both host determinants  and virus determinants . Some good examples are provided by the New molecular approaches have rapidly expanded the list of enteric viruses  for which pathology is currently less evident Cardiovirus of the picornavirus family. SAFV-3, one of its genotypes, shows a high seroprevalence on three different continents, indicating wide-spread presence of infection among humans. Infection by SAFV-3 is most likely acquired early in life. Infection may pass without symptoms but the full clinical and/or biological spectrum remains still to be established.In conclusion, the SAFVs, are human viruses belonging to the genus Fecal and respiratory samples are routinely cultured on human embryonic lung fibroblasts (HELF), tertiary monkey kidney cells (tMK) and Hep-2 cells. Isolates with a cytopathic effect (CPE) suggestive for a picornavirus are tested for acid- and chloroform resistance. If so, the isolates are typed using enterovirus typing serum pools provided by the National Institute for Public Health and Environment, Bilthoven, The Netherlands (RIVM) The virus was partially purified by centrifugation through a layer of 30% (w/w) sucrose in PBS for 3 h at 250,000×G, 4°C. RNA was isolated from the pellet using Trizol-B (Invitrogen) according to the protocol of the manufacturer and amplified using a random PCR method Nucleotide and protein sequences of the following cardiovirus genomes were obtained from GenBank (accession numbers): TRV NGS910 Japan 1991 (AB090161); TRV-1 USA 2006 (EU815052); VHEV (M80888 and EU723237); TMEV DA (M20301); TMEV GDVII (NC_001366); SAFV-1 USA 1981 (NC_009448); SAFV-2 D2229 2004 (EU681176); SAFV-2 UC-1 2005 (EU376394); SAFV-2 Can 2006 (AM922293); SAFV-2 BR118 2006 (EU681177); SAFV-3 D2273 2004 (EU681178); SAFV-3 D2223 2004 (EU681179); SAFV-3 NL2007 (FM207487). Sequence alignment was performed using ClustalW A virus stock of SAFV-3 was produced on HeLa cells, aliquoted, and titrated by end-point titration on HeLa cells. For growth curves, subconfluent monolayer cells were infected with virus at a multiplicity of infection (MOI) of 10 for 30 min at room temperature. Cells were grown at 36°C for 0, 8, 24, 48 or 72 h. At these time points, viruses were released by three cycles of freezing and thawing. Virus titers were determined by end-point titration on HeLa cells as previously described Virus neutralisation (VN) assays were performed on human epithelial cervical carcinoma (HeLa) cells in a microtiter assay as described for enteroviruses A total of 375 serum specimens were tested, belonging to three categories: a) routine viral diagnostic samples (n = 210); b) serum samples from 30 Finnish children collected for a surveillance study on antibodies against polioviruses; c) samples from 135 individuals living in two African countries and two different areas in Indonesia collected for studies on malaria. Permission for anonymous testing of the samples fulfilled the terms of the ethical code “Goed Gebruik” of the Netherlands Federation of Medical Scientific Societies (Federa).Rodent serum samples have been provided by QM Diagnostics BV. Pooled rodent sera have been used which were tested negative for a viral annual test panel recommended by FELASA . Sera were derived from rats, mice, hamsters, guinea pigs, and rabbits. In addition, pooled sera from rats positive for TMEV and sera from mice containing high titer antibodies to TMEV or EMCV confirmed positive by ELISA and immunoblot were tested.Figure S1Structure and alignment of SAFV CRE. (A) Secondary structure of the SAFV-3(NL2007) CRE as predicted by MFOLD. (B) Alignment of cardiovirus VP2 sequences ) corresponding to the CRE region. Nucleotides involved in stem formation are indicated.(0.07 MB PDF)Click here for additional data file.Figure S2SAFV L and L* proteins. (A) Sequence alignment of the cardiovirus leader proteins. Cysteine and histidine residues involved in the formation of the zinc-finger are shaded. The zinc-finger-, acidic-, and the serine/threonine-rich domains are indicated. The EMCV L protein is phosphorylated at threonine-47. This phosphorylation site fails in TMEV and VHEV. It is importantly to note that although all SAFVs contain a threonine at the corresponding position, the identity of the surrounding aa render it an unfavorable phosphorylation site. (B) Sequence alignment of putative cardiovirus L* proteins. The first 75 aa from the +1 frame shifted ORF are shown. Stop codons are depicted as *. Note that the L* proteins of the TMEV-GDVII strain and the SAFVs contain an ACG start codon.(0.07 MB PDF)Click here for additional data file.Table S1Nucleotide and amino acid (in parenthesis) identity of SAFV-3(NL2007) UTRs and proteins to other Theiloviruses from which full-length sequences or polyprotein coding regions are known.(0.09 MB PDF)Click here for additional data file.


A detailed patient-by-patient costing analysis, based on case records for 253 patients diagnosed in 1993, reveals that the mean 4-year diagnosis and management costs amounted to £6150 and £5668 for non-small cell and small cell lung cancer respectively. These costs are lower than those identified in Canadian studies, the difference being explained by the use of a simulated costing methodology in these studies, lower unit costs and less aggressive interventions. © 1999 Cancer Research Campaign