Myelosuppressive chemotherapy can lead to dose-limiting febrile neutropenia. Prophylactic use of recombinant human G-CSF such as daily filgrastim and once-per-cycle pegfilgrastim may reduce the incidence of febrile neutropenia. This comparative study examined the effect of pegfilgrastim versus daily filgrastim on the risk of hospitalization.This retrospective United States claims analysis utilized 2004–2009 data for filgrastim- and pegfilgrastim-treated patients receiving chemotherapy for non-Hodgkin’s lymphoma (NHL) or breast, lung, ovarian, or colorectal cancers. Cycles in which pegfilgrastim or filgrastim was administered within 5 days from initiation of chemotherapy (considered to represent prophylaxis) were pooled for analysis. Neutropenia-related hospitalization and other healthcare encounters were defined with a “narrow” criterion for claims with an ICD-9 code for neutropenia and with a “broad” criterion for claims with an ICD-9 code for neutropenia, fever, or infection. Odds ratios (OR) for hospitalization and 95% confidence intervals (CI) were estimated by generalized estimating equation (GEE) models and adjusted for patient, tumor, and treatment characteristics. Per-cycle healthcare utilization and costs were examined for cycles with pegfilgrastim or filgrastim prophylaxis.We identified 3,535 patients receiving G-CSF prophylaxis, representing 12,056 chemotherapy cycles . The mean duration of filgrastim prophylaxis in the sample was 4.8 days. The mean duration of pegfilgrastim prophylaxis in the sample was 1.0 day, consistent with the recommended dosage of pegfilgrastim - a single injection once per chemotherapy cycle. Cycles with prophylactic pegfilgrastim were associated with a decreased risk of neutropenia-related hospitalization  and all-cause hospitalization  versus cycles with prophylactic filgrastim. For neutropenia-related utilization by setting of care, there were more ambulatory visits and hospitalizations per cycle associated with filgrastim prophylaxis than with pegfilgrastim prophylaxis. Mean per-cycle neutropenia-related costs were also higher with prophylactic filgrastim than with prophylactic pegfilgrastim.In this comparative effectiveness study, pegfilgrastim prophylaxis was associated with a reduced risk of neutropenia-related or all-cause hospitalization relative to filgrastim prophylaxis. Patients receiving myelosuppressive chemotherapy are at risk of developing febrile neutropenia, a major dose-limiting toxicity of systemic chemotherapy associated with hospitalization, use of intravenous antibiotics, and significant morbidity, mortality, and costs -10. RecoFilgrastim is recommended to be administered at a recommended starting dose of 5 mcg/kg per day until neutrophil recovery occurs after the expected chemotherapy-induced nadir. Its short circulating half-life (~3.5 hours) necessitates that it be given daily. The efficacy of filgrastim depends on the number of days it is administered. Randomized comparative clinical trials have shown that patients treated with a mean of 10–11 filgrastim doses per chemotherapy cycle reported a similar decrease in the duration of severe neutropenia as patients treated with pegfilgrastim once per chemotherapy cycle -18. In cNeutropenic complications following chemotherapy contribute significantly to the costs of cancer care. During 1989–2007, the number of neutropenia-related hospitalizations among cancer patients in the United States was estimated to be approximately 57,000–103,000 per year [The primary objective of the current study was to determine whether a difference in the risk of neutropenia-related and all-cause hospitalization between chemotherapy cycles associated with filgrastim prophylaxis and cycles with pegfilgrastim prophylaxis. This study from a United States claims database included data from January 2004 through February 2009 regarding filgrastim and pegfilgrastim administration patterns and related clinical outcomes. Additionally, economic data in the form of comparative healthcare utilization and costs results are described.This study was a retrospective United States claims analysis using data from health plans affiliated with OptumInsight . This national database contains both medical and pharmacy claims with linked enrollment information data beginning in 1993. As of 2008, medical and pharmacy benefit coverage information was available for over 14 million individuals.All patient-identifying information was either encrypted or removed from the study database prior to its release to the study investigators. The study database does not contain any Protected Health Information and is fully compliant with the Health Insurance Portability and Accountability Act (HIPAA) of 1996 and federal guidance on Public Welfare and the Protection of Human Subjects ,25. As pOutcomes, including administration patterns, neutropenia-related and all-cause hospitalization rates, and utilization and cost data, were obtained for both filgrastim- and pegfilgrastim-treated patients receiving chemotherapy for non-Hodgkin’s lymphoma (NHL), breast cancer, lung cancer, ovarian cancer, or colorectal cancer.Patients with chemotherapy medical claims between January 1, 2005 and February 28, 2009 were studied. The year prior to the index date was used to determine whether patients met inclusion/exclusion criteria and to provide demographic and patient characteristic data. The date of the first chemotherapy claim of an eligible patient was deemed the index date. Patients were included if they had two or more medical claims ( ≥ 7 days apart) with ICD-9 code(s) for NHL, breast cancer, lung cancer, ovarian cancer, or colorectal cancer from 30 days prior to the index date up to 30 days after the index date, and one or more claim for filgrastim or pegfilgrastim (not both) during the chemotherapy course. Claims from laboratories, diagnostic testing centers, or any diagnostic tests were not considered when identifying cancer patients, as well as claims with “rule-out” codes . Patients were excluded if they had less than 1 year of continuous eligibility preceding the index date; any claim for chemotherapy during the 1 year prior to the index date; one or more medical claims for bone marrow or stem cell transplant; claims indicating sargramostim use; claims for services provided in skilled nursing facility or hospice services; or if they had codes for more than one type of primary cancer. Patients with metastatic disease were not specifically excluded.Besides tumor type and use of pegfilgrastim/filgrastim, data collected also included demographic characteristics, comorbid conditions, cancer treatment history, and chemotherapy agents received in each chemotherapy cycle. The first eligible chemotherapy course for each patient after January 1, 2005 was used in this analysis. Each chemotherapy course may include several cycles. The first chemotherapy course began on the index date and ended with any of the following, whichever came first: 1) the absence of any chemotherapy claims within the 60 days after a chemotherapy claim ; 2) the end of insurance eligibility or study period; or 3) the initiation of radiation therapy. Chemotherapy cycles in the course were defined to identify unique cycles of interest, and were excluded if two chemotherapy claims had less than 20 days between them or if there were chemotherapy claims from days 7–19 of a cycle . ChemothFor both filgrastim and pegfilgrastim, use was categorized as prophylactic  or delayed (initiated after day 5 of a cycle) ,21. The Healthcare utilization and costs were calculated for each cycle. Total all-cause costs were calculated as the costs associated with all medical and pharmacy claims during the cycle. Physician fees, chemotherapy costs, and G-CSF costs were all included in the total all-cause cost measure. Additionally, all-cause utilization and costs were calculated separately for emergency room (ER) visits, hospitalizations, and ambulatory care visits . Hospitalization length of stay was another examined utilization outcome. Drugs delivered as part of medical benefits  were included in the costs for each setting of care: ER, hospital inpatient, and ambulatory care. Retail pharmacy costs were calculated as the cost for all prescription claims and considered as a component of total costs in the ambulatory care setting. Neutropenia-related utilization and costs were calculated from claims associated with neutropenia. The costs represented the reimbursed amount paid by the patient and insurer. Only direct costs for services covered under the patient’s insurance benefit were included in this study.Descriptive statistics regarding patient characteristics and cycle characteristics (in terms of chemotherapy agents received and G-CSF administration patterns) were calculated for filgrastim and pegfilgrastim separately. Additionally, descriptive statistics were calculated for healthcare utilization, costs, and length of hospital stay during cycles with filgrastim or pegfilgrastim prophylaxis. Statistical tests were used based on the distribution of the data . Odds ratios (OR) and 95% confidence intervals (CI) for hospitalization in cycles with pegfilgrastim prophylaxis compared to cycles with filgrastim prophylaxis were estimated by generalized estimating equation (GEE) models. The GEE models included up to the ninth chemotherapy cycle for each patient. This restriction was included because failure to do so resulted in GEE model estimation that failed to converge. A binomial distribution with log link was specified for all GEEs, and the models were fit using an exchangeable correlation structure. To control for possible confounding between G-CSF agent and outcomes, ORs were adjusted for patient age and sex, cancer type, myelotoxicity of chemotherapy, chronic comorbidities (as assessed with the Quan-Charlson comorbidity index ), and hiSensitivity analyses were performed to determine whether other factors could affect the results. First, we examined the impact of excluding filgrastim cycles with a shorter duration  of filgrastim prophylaxis. Second, as the exclusion of patients with evidence of more than one primary cancer could inappropriately exclude patients with only one primary cancer , that criterion was removed.The initial population from the claims database contained 151,118 patients receiving chemotherapy. After applying the inclusion and exclusion criteria, the eligible population included 3,535 patients with breast cancer, lung cancer, NHL, ovarian cancer, or colorectal cancer receiving G-CSF prophylaxis. These patients represented 12,056 cycles during which G-CSF was delivered prophylactically, including 373 filgrastim cycles and 11,683 pegfilgrastim cycles  per cycle was 1.3% (95% CI: 0.4%–3.1%) with prophylactic filgrastim and 0.6% (95% CI: 0.5%–0.7%) with prophylactic pegfilgrastim (P = 0.063); neutropenia-related hospitalization (broad definition) per cycle also occurred at a higher incidence with prophylactic filgrastim than with prophylactic pegfilgrastim ; a higher incidence with prophylactic filgrastim than with prophylactic pegfilgrastim in all-cause hospitalizations per cycle was observed as well .When compared with cycles in which filgrastim was prophylactically used, cycles in which prophylactic pegfilgrastim was used were associated with a reduction in the risk of all-cause hospitalizations  and neutropenia-related hospitalization , after controlling for patient, disease, and treatment characteristics in GEE models  and all causes Table . BecauseFor neutropenia-related utilization per cycle for all cycles (including cycles without neutropenia-related utilization), there were more ambulatory visits  and hospitalizations  with filgrastim than with pegfilgrastim (mean ER visits were 0 for both groups). In the subset of filgrastim cycles in which neutropenia-related hospitalization occurred , an average of 1.4 neutropenia-related hospitalizations occurred per cycle, with mean length of stay of 12.6 days. In the subset of pegfilgrastim cycles in which neutropenia-related hospitalization occurred , an average of 1.0 neutropenia-related hospitalization occurred per cycle, lasting a mean of 7.0 days each. Among all filgrastim cycles, 39.7% were associated with at least one neutropenia-related ambulatory visit; the corresponding percentage among all pegfilgrastim cycles was 28.0%. During the subset of cycles in which these neutropenia-related ambulatory visits occurred, the mean number of neutropenia-related visits was 5.3 for filgrastim cycles and 1.3 for pegfilgrastim cycles. All-cause resource utilization showed similar trends as that indicated for neutropenia-related results, and specific all-cause healthcare utilization results can be seen in Table Per-cycle costs were also examined by setting of care: ambulatory care, ER, and inpatient hospitalizations Figure . Mean neThe combined mean costs per cycle due to all cause for all cycles were fairly similar for the two G-CSF molecules, with $9,575 for filgrastim and $9,786 for pegfilgrastim (P = 0.756). Compared to pegfilgrastim, there were lower ambulatory care costs for filgrastim  and greater hospitalization costs for filgrastim .In this retrospective United States claims analysis, use of prophylactic pegfilgrastim was associated with a decreased risk of neutropenia-related hospitalization  and all-cause hospitalization  when compared to that seen with the use of prophylactic filgrastim. Patients who received filgrastim prophylaxis in this study had a mean 4.8 days of prophylaxis, which is much shorter than the duration which has been demonstrated to be non-inferior to pegfilgrastim prophylaxis in clinical trials -18. Thes–2005) [In a previous study that used a large managed care claims database in the United States, Weycker et al. performed a retrospective cohort analysis of cancer patients who received filgrastim or pegfilgrastim during their first course of chemotherapy (2003–2005) . Both al–2005) ,23. The –2005) -18; in t–2005) -23.One distinct aspect of this study is that comparative filgrastim and pegfilgrastim costs and resource utilization data were reported, including details such as the number of hospitalizations, ambulatory and ER visits, and per-cycle costs. Comparative studies assessing cost and resource utilization of filgrastim and pegfilgrastim in the United States are limited. A retrospective single-time-point survey conducted by Fortner et al. assessed the human resource costs required for administering filgrastim or pegfilgrastim . They coIn this study utilizing a large claims database, all-cause costs were roughly equivalent for filgrastim and pegfilgrastim on a per-cycle basis, with more spent on hospitalizations with filgrastim and more spent on ambulatory care with pegfilgrastim. It is interesting to note that although pegfilgrastim cycles had greater costs for ambulatory care, there were proportionally more ambulatory visits for filgrastim cycles. The increased all-cause per-cycle cost of pegfilgrastim ambulatory care may reflect the greater drug costs of pegfilgrastim compared to filgrastim, especially when fewer than the recommended number of filgrastim doses were administered. Neutropenia-related costs were greater for filgrastim than pegfilgrastim because of the greater costs of both inpatient and ambulatory care during filgrastim cycles. Overall, these data suggest that the greater drug costs with pegfilgrastim are offset by decreased hospitalization costs.There are several sources of bias and limitations inherent in the study design that could influence interpretation of these results. As this was a database of a large employment-sponsored managed care population, patients would most likely be in the 18–64 year age range. Thus, the effects of G-CSF on outcomes in the population of patients aged 65 years or above were not fully captured. Additionally, the data are dependent on the accuracy of claims coding and hence contain any errors or omissions that occurred during that coding. By using the broad criteria for neutropenia, as well as all-cause utilization, we were able to more accurately provide upper limits for our estimates. It should be noted that this study compared risk between treatment cohorts. The potential under-coding and mistakes in coding of febrile neutropenia are unlikely to be associated with G-CSF selection and thus do not affect the estimates of interest . Sample size is another issue that affects the statistical power of our estimates, as there were fewer than 400 cycles with prophylactic filgrastim use. Another source of bias is the assumption that G-CSF administration by day 5 of a cycle represents prophylaxis rather than treatment. Although this definition has been used in other studies ,21, its It is unclear whether this study adequately captured the various known patient, disease, and treatment characteristics that are risk factors for developing febrile neutropenia ,12. The The use of per-cycle analyses for utilization and costs has the disadvantage of not capturing costs associated with cycles in which G-CSF was not administered. However, per-patient analyses would not allow us to temporally associate use of either G-CSF with utilization. For instance, in a per-patient analysis, a patient hospitalized in cycle 1 who subsequently received G-CSF in cycle 2 would have those two events associated, when clearly the hospitalization was independent of any effects from G-CSF. Another disadvantage of per-patient analyses is that while on average, each patient who received filgrastim received it for a mean of 2.3 cycles, while each patient who received pegfilgrastim received it for a mean of 3.5 cycles. The accompanying drug costs would make it difficult to discern any possible effect of either medication on all-cause medical costs.In conclusion, the results of this claims analysis indicate that prophylactic use of filgrastim as compared with pegfilgrastim is associated with an increased risk of hospitalization from all causes as well as neutropenia-related causes. Results from this analysis and others indicate that other factors influence the risk of hospitalization, including comorbidities, history of anemia, age, and presence of metastatic disease ,11,12,29This retrospective comparative effectiveness study used claims data to examine prophylactic use of filgrastim and pegfilgrastim in cancer patients receiving chemotherapy. The key finding of the study is that pegfilgrastim prophylaxis was associated with a reduced risk of hospitalization due to neutropenia or all-cause.In addition to the relationships with Amgen Inc. described in the Acknowledgements, Henry Henk and Laura Becker are employees of OptumInsight, while Victoria Chia, Sejal Badre, Xiaoyan Li, and Robert Deeter are stockholders of Amgen Inc.All authors contributed to study design and analysis and interpretation of data. HH and LB were primarily responsible for data acquisition. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/11/prepubAppendix - Bacterial and Fungal Infections.Click here for file


Cryptococcus neoformans and Cryptococcus gattii, is particularly problematic because this fungal infection occurs in immunocompromised and apparently immunocompetent individuals. It has been largely accepted that Cryptococcus species are recognized by cellular receptors and that Th1-type immune responses play an important role in defense mechanisms against the yeast. However, the interaction between the yeast and host tissue varies depending on the characteristics of the yeast and the immune status of the host. To gain a better understanding of the pathophysiology of cryptococcosis, we wish to emphasize the usefulness of histopathological examinations, because it allowed more detailed information of an extremely complex interaction between the causative yeasts and tissue response. In the present review, we describe the pathophysiology of cryptococcosis as largely revealed in our previous histopathological investigations of the experimental infection.Invasive fungal infections, particularly those considered opportunistic, have become a common and significant complication of procedures performed in advanced contemporary medicine. Among such infections, cryptococcosis, which is usually caused by infection with  Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). C. neoformans is widely distributed throughout the world [C. gattii has been limited in tropical and subtropical regions [C. gattii infection in a temperate region of British Columbia in 1999, which expanded towards US Pacific Northwest and Japan [The increasing use of invasive monitoring and aggressive therapeutic technologies in developed countries has resulted in improved survival of individuals with life-threatening diseases –3. Howevhe world , whereas regions . Howevernd Japan –10.Cryptococcus species are recognized by cellular receptors and that Th1-type immune responses play an important role in defense mechanisms against the yeast [Recent molecular biological investigations have contributed significantly toward an elucidation of the pathogenesis of the yeast. It has been largely accepted that he yeast . Howeverhe yeast , 13. Thehe yeast . In the C. neoformans TIMM 0362, a relatively high-virulence and thus high-mortality strain, or TIMM 0372, a relatively low-virulence and thus low-mortality strain, via the intranasal route of infection [Primary foci of a cryptococcal infection tend to be established in the lungs before disseminating to the central nervous system . To detenfection . C. neoformans TIMM 0362 showed yeast cell proliferation in the alveoli and a lesser macrophage response (C. neoformans TIMM 0372 showed numerous yeast-containing multinucleated giant cells (MGCs) due to the prominent macrophage response  class II molecules on the surface of the antigen-presenting cells (APCs) and CD4-positive T lymphocytes plays a central role in regulating the cell-mediated immune response against infection with the Cryptococcus species [C. neoformans TIMM 0362 and TIMM 0372 by immunohistochemical examination. As a result, the former showed no expression of MHC class II IAd molecule, and the later expressed this molecule , and another is term of infection (7 days versus 14 days). Since we were able to observe histopathological findings of the lesion just before death of the infected mice, macrophage response could be amplified in our study. In addition, Cheng et al. employed flow-cytometric analysis to count the number of macrophages that is superior to manual counting of the cell, but it cannot count the number of macrophages within MGC, accurately. Since macrophage fused together to form a MGC, the flow-cytometric analysis was unable to accurately assess the macrophage response.On the other hand, Cheng et al. reported that no significant difference was found in number of macrophages between C. gattii and C. neoformans infections. Namely, both extensive proliferation of the yeast in alveoli during C. gattii R265 infection and macrophage response to intruding C. neoformans H99 would cause respiratory failure of the mice. Despite the same mortality rates between mice infected with C. gattii R265 and C. neoformans H99 was found, the pathophysiology leading to death between C. gattii and C. neoformans infection was different, which was only revealed by histopathological examination. We therefore wish to emphasize the usefulness of the yield of the histopathological examination for experimental animal model.Consequently, there is an extensively different pathophysiology leading to death between C. gattii is characterized by its ability to induce high pulmonary structural alteration [C. gattii (TIMM 4097 and TIMM 4901 to 4906) [ C. gattii TIMM 4097, TIMM 4901 to 4906 were isolated from Japanese zoo-bred koalas. Serotypes of the strain were confirmed using slide agglutination tests according to the manufacturer's instructions . All animal studies were performed in accordance with the guidelines and permission of the animal experiment care committee of the Teikyo University. On day 100 after infection, the cumulative mortality rates in mice infected with C. gattii TIMM 4097 was 10/10 (100.0%), whereas the rates in mice infected with C. gattii TIMM 4901 to 4906 were 0/10 (0.0%). According to Kaplan-Meier survival analysis with a log-rank significance test, the mortality rate in mice infected with C. gattii TIMM 4097 was significantly higher than that of mice infected with C. gattii TIMM 4901 to 4906. We found that teration . To gainto 4906) , 29. C. C. gattii, including growth rate, capsule thickness, melanin production, and hydrolase  activity. Despite the significant differences in mortality rate among mice infected with the different strains, no significant differences were found among the strains regarding any of the characteristics examined, except for growth rate. The results showed that only the growth rate suggested a correlation with the mortality rate of infected mice.To elucidate factors affecting virulence of the yeast, we examined biological characteristics of seven strains of C. gattii strain plays a strong role in its virulence [C. gattii using multilocus sequence typing (MLST) analysis in accordance with the consensus C. gattii typing scheme established by the Cryptococcal Working Group I of the International Society for Human and Animal Mycology (ISHAM) [CAP59, GPD1, LAC1, PLB1, SOD1, URA5, and IGS1. Although the mortality rate of mice infected with C. gattii TIMM 4907 was significantly higher than that of mice infected with C. gattii TIMM 4901 to 4906, MLST analysis revealed that the molecular type of all seven strains was VG I  , which iwas VG I , indicatC. gattii strains of VG I isolated from Japanese zoo-bred koalas, the mortality rate of mice infected with C. gattii TIMM 4097 was significantly higher than that of mice infected with other six C. gattii strains. Therefore, we conducted detailed histopathological examination to gain an insight into the pathophysiology underlying the virulence. As the C. gattii TIMM 4901 to 4906 strains were found to have the same phenotypic expression, molecular type, and mortality rate in infected mice, C. gattii TIMM 4903 was randomly selected as a representative type for further examination and comparison with C. gattii TIMM 4097. Time-dependent histopathological examination revealed that pulmonary sections obtained from mice infected with C. gattii TIMM 4097 showed eccentric enlargement of alveoli containing prominent proliferation of yeast cells with disease progression  tend to induce greater alteration of lung structure than infection with C. gattii TIMM 4903 (a low-virulence strain). To investigate the hypothesis that C. gattii TIMM 4097 tends to reside in the alveoli, the extent to which the yeast cells filled the bronchi was determined by semiquantitative assessment. All bronchi of the pulmonary PAS-stained sections were observed, and the bronchi were classified as “empty,” “partially full,” or “completely full” based on the degree to which the bronchi were filled with yeast cells. Results revealed that the ratio of “completely full” bronchi in mice infected with C. gattii TIMM 4903 was significantly higher than that of mice infected with C. gattii TIMM 4097 throughout the observation period . All the microarray data are deposited in GEO (accession number GSE48595). The microarray analysis detected the expression of 45,101 genes (P ≤ 0 to <0.04), “marginal” (P ≤ 0.04 to <0.06), or “absent” (P ≤ 0.06 to <0.5). Gene expression that contained the “absent” flag  was excluded, and the expression of 5,920 genes was extracted  analysis of the stored genes was conducted using the Biological Networks Gene Ontology tool  to identify statistically overrepresented GO categories among the biological data. For the 184 genes upregulated only in the lungs of mice infected with C. gattii TIMM 4097, 163 terms related to molecular functioning were detected, and GO terms related to binding accounted for about 75% of all terms (A microarray assay with gene ontology analysis was also performed with reference to the previous investigation . Gene ex01 genes  and the xtracted . As refeIMM 4097 . To examll terms . C. gattii TIMM 4097 rigidly resides in the alveolar space and that this may cause alveolar expansion, whereas C. gattii TIMM 4903 is likely to be washed out from the alveoli and move into the central side of the respiratory system, as indicated by observation of little change in lung structure of mice infected with this strain. These differences may have arisen from the different capacity of the two strains to adhere onto the alveolar epithelium and is partly confirmed by our previous study.Our previous investigations suggested that The defense mechanism against cryptococci may vary among individuals, depending on the agents, tissues involved, and immune status of the host. Given these considerations, we wish to emphasize the usefulness of histopathological examinations, because it allowed more detailed information of an extremely complex interaction between the causative microbes and tissue response.


In 2011, an unidentified disease in cattle was reported in Germany and the Netherlands. Clinical signs included fever, decreased milk production, and diarrhea. Metagenomic analysis identified a novel orthobunyavirus, which subsequently was isolated from blood of affected animals. Surveillance was initiated to test malformed newborn animals in the affected region. In summer and autumn 2011, farmers and veterinarians in North Rhine-Westphalia, Germany, and in the Netherlands reported to the animal health services, local diagnostic laboratories, and national research institutes an unidentified disease in dairy cattle with a short period of clear clinical signs, including fever, decreased milk production, and diarrhea. All classical endemic and emerging viruses, such as pestiviruses, bovine herpesvirus type 1, foot-and-mouth disease virus, bluetongue virus, epizootic hemorrhagic disease virus, Rift Valley fever virus, and bovine ephemeral fever virus, could be excluded as the causative agent. To identify the cause of the disease, we analyzed blood samples from affected cattle.www.insdc.org) databases .On a farm near the city of Schmallenberg  for codons. Phylogenetic relationship was assessed by using the neighbor-joining method based on a Tamura 3-parameter model and bootstrap analysis  as implemented in MEGA5  was aligned with sequences of the Simbu, Bunyamwera, and California serogroups by using ClustalW  values of 24–35. All farms with cattle that tested positive were sampled in September, October, or November and are located within the federal state of North Rhine-Westphalia. Most farms are in close proximity to the border with the Netherlands  (primers and probes are available on request) was used to test additional samples from affected cattle farms. Twelve samples, mainly from adult cattle from 6 different farms, were positive for the novel virus, with cycle threshold (Cherlands . The latCulicoides variipennis larvae cells (KC cells)  3 calves, ≈9 months of age, were inoculated directly with blood that was PCR positive for the novel virus from 4 different cattle  or with the initial KC cell isolate described above (1 mL subcutaneously and 4 mL intravenously). All inoculated animals became infected and had positive PCR results 2–5 days postinoculation (dpi), with the lowest Cat 4 dpi , panel BThe detection of a novel orthobunyavirus in cattle in Germany  demonstrates the power of a metagenomic approach to discovering emerging pathogens. Specific and sensitive RT-qPCRs could be developed quickly and used in analyzing infected herds.The role of the virus in the disease needs to be further investigated. However, the clinical signs in 2 of the inoculated animals, together with virus detection in samples of diseased animals in Germany and the Netherlands (Concern exists about the congenital defects the virus might induce in newborn calves, goats, and lambs during the next months. Therefore, surveillance has been initiated to test all malformed animals in the affected region. Some members of the Simbu serogroup, e.g., Oropouche virus, are zoonotic. However, because of the close relationship to Shamonda virus and the absence of reports of clinical signs in humans, the risk to humans currently is assessed as very low to negligible. Nevertheless, clinical and serologic surveillance in humans should be conducted in regions with infected animals to update the risk assessments.


Eidolon helvum, an old world fruit bat species, and henipavirus-like nucleic acid has been detected in faecal samples from E. helvum in Ghana. The initial outbreak of NiV in Malaysia led to over 265 human encephalitis cases, including 105 deaths, with infected pigs acting as amplifier hosts for NiV during the outbreak. We detected non-neutralizing antibodies against viruses of the genus Henipavirus in approximately 5% of pig sera (N = 97) tested in Ghana, but not in a small sample of other domestic species sampled under a E. helvum roost. Although we did not detect neutralizing antibody, our results suggest prior exposure of the Ghana pig population to henipavirus(es). Because a wide diversity of henipavirus-like nucleic acid sequences have been found in Ghanaian E. helvum, we hypothesise that these pigs might have been infected by henipavirus(es) sufficiently divergent enough from HeVor NiV to produce cross-reactive, but not cross-neutralizing antibodies to HeV or NiV.Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), have Pteropid bats as their known natural reservoirs. Antibodies against henipaviruses have been found in  Henipavirus in the family Paramyxoviridae is comprised of Hendra (HeV) and Nipah (NiV) viruses. These viruses use bats of the family Pteropodidae as their natural reservoir hosts The genus Eidolon helvum bats in Ghana, West Africa Eidolon helvum roosts in large colonies, reaching several million in number, and has a wide distribution across the African continent. Those findings thus extended the range of henipaviruses from Asia and Australasia to Africa. A subsequent study found henipavirus-like nucleic acid in faecal samples from E. helvum in Ghana We previously reported serological evidence for henipavirus infection in E. helvum colony  resides for approximately 6 months during each dry season.To test this hypothesis, we screened a selection of domestic animal sera from animals within the grounds of the 37 Military Hospital, Accra, Ghana, where a large Trypanosoma study undertaken at the Veterinary Services Laboratory, Ghana. Both villages are in the Suhum/Kraboa/Coalta district, Eastern Region, about 70 km north of Accra. Sample numbers P1–48 and P50 were from 25 households in Kwesikonfo  and P52–64 and P66–100 from 11 households in Zorh (N 5°59 W 0°21). The bat-pig contact history was unknown. However, villages contain fruit trees and the pigs were housed in open pens (1–10/pen), with some running free during the day, and bats of numerous species have been caught by the authors foraging in other villages in the region Ethical approval for this project (WLE/0467) was received from the Zoological Society of London Ethics Committee and locally from the Ghanaian Veterinary Services Directorate. Serum samples were collected in June 2007 from 2 cats, 2 dogs, 10 sheep and 15 goats. In addition, 97 pig samples were available from 2 villages, collected as part of a NiV and sGHeV-coupled microsphere subsets were pre-mixed and incubated with sera, followed by incubation with biotinylated Protein A/G and streptavidin–phycoerythrin. Antibodies bound to the sGNiV or sGHeV coated beads, which are spectrally distinct, are quantified by the fluorescence emitted by phycoerythrin. This is read as the median fluorescence intensity (M.F.I.). Gamma-irradiated positive pig and cat sera controls from naturally or experimentally infected animals, and negative controls from each species were used. Putative positive sera, with M.F.I. titres 3-fold above the negative sera M.F.I., were then tested using a Luminex ephrin-B2 receptor blocking (inhibition) assay and by virus neutralization tests (VNTs) All sera were tested for antibodies binding to both a HeV and NiV recombinant soluble G glycoprotein (sG) using a Luminex® multiplexed binding assay, as described previously Finally, 7 pig sera with high Luminex binding titres, and 1 with a low Luminex binding titre (P17) were tested by western blot (WB). The WB was performed using purified recombinant Nipah virus nucleocapsid (N) protein produced as previously described One goat (G) and one sheep (S) sample each had M.F.I. titres well above 3 times background for NiV and HeV respectively. G15′s titre was 584 M.F.I. against NiV (>3 times mean negative sera M.F.I. of 62), but only produced a titre of 71.5 M.F.I. against HeV (negative sera M.F.I. 62). Sheep S7′s M.F.I. titre was 521 against HeV (>3 times mean negative sera M.F.I. of 81), but only 86 M.F.I. against NiV (negative sera M.F.I. 66) see . NeitherAll sera tested for inhibition  were tested by VNTs, but no sera tested positive using VNTs. However, one (P92) of the seven binding-antibody positive pig sera tested positive by WB against the N protein , thus confirming non-neutralizing antibody against another NiV protein. No further sample was available for P29 for testing by WB. The additional WB staining seen in all samples except P92 is thought to be non-specific background staining.Our results suggest evidence of prior exposure of the Ghana pig population to henipavirus(es). The wide diversity of henipavirus-like nucleic acid sequences that have been found in Ghanaian bats E. coli proteins. However, given binding antibody had previously been demonstrated against sG NiV proteins in this sample of pig sera, we believe this adds further evidence of infection by a NiV or Nipah-related virus in these populations.Luminex binding assay has been demonstrated to give preferentially higher M.F.I. for the virus that induced antibody from field sera of NiV infected pigs from the 1998–1999 NiV outbreak in Malaysia and Singapore, and HeV infected horses from the 1994 HeV outbreak The mechanisms of virus neutralization are complex and could involve more antigenic sites than those required for simple receptor binding or inhibition. The failure of finding VNT positive sera therefore may be due to the divergent nature of the viruses inducing antibody. As has been shown in studies on bats in Ghana, only a relatively small proportion of those seropositive by Luminex assay were VNT positive, and subsequent studies by others have shown related but divergent henipavirus sequences from the same bat species. Furthermore, previous studies have shown the NiV antibody positive pig sera produced lower titres than human , bat (HeV) and horse (HeV) positive sera using all three  assays Laboratory studies suggest viral replication in bats is limited E. helvum frequently roosts in urban areas, is a source of bushmeat Finally,


We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus “spillover” into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves. Methods to reduce the transmission of HIV-1 are hindered by a lack of information regarding early viral dynamics and evolution in the male genital tract. In the present study, we show that SIV in the blood and genital tract are homogeneous during early infection, indicating facile virus gene flow between these compartments. Importantly, the coincidence of the resolution of primary viremia with the decreased virus levels in genital secretions suggest that the dramatic fall in virus replication during early infection underlies the development of viral compartmentalization. Our demonstration of early virus compartmentalization in the male genital tract has important implications for the understanding of early events leading to infection of the male genital tract and the nature of the transmitted virus during primary retrovirus infection. An understanding of HIV-1 biology in the male genital tract will be central to understanding the transmission of this virus. Since HIV-1 is transmitted predominantly by sexual contact HIV-1 exists as a diverse population of related genetic variants that segregate into subpopulations within anatomic compartments of infected individuals Importantly, the male genital tract serves as a reservoir for HIV-1 that has been shown by some investigators to harbor virus that is distinct at the sequence level from virus found in the blood env) sequences in the blood and semen at the peak and set point of SIV replication in rhesus monkeys. We observed a striking progression toward increasing viral compartmentalization over time following infection, raising the possibility that the abrupt reduction in virus replication that occurred in these monkeys after the resolution of peak viremia may contribute to a restriction in viral gene flow in and out of their genital tracts.In the present study we sought to define the dynamics of SIV compartmentalization in semen during primary infection in rhesus monkeys. We accomplished this by analyzing SIV envelope  was used to generate 789 env amplicons for sequencing and subsequent phylogenetic analysis. Sequence data were generated from 2 time points post-challenge, at week 2 (peak virus replication) and week 16 (virus set point).To determine whether virus in the semen and peripheral blood were compartmentalized following infection, we assessed the phylogenetic relationship between semen- and blood-derived SIV Viral RNA levels were comparable for each group, and we detected no significant differences in week-2 and week-16 blood or seminal plasma viral loads between vaccinees and controls. Similarly, no significant differences were detected between animals included in this study and animals excluded as a result of insufficient sequence data (Wilcoxon P>0.01) as described env sequences were first generated from both the blood and semen of 14 rhesus monkeys (7 vaccinated and 7 control) from specimens obtained 2 weeks following infection. The genetic diversity of the virus from each anatomic compartment of each monkey was calculated as the average pair-wise genetic distance of SIV env within each semen or blood population from the inoculum consensus. Data from the vaccinated and control animals were analyzed independently. Since these represented analyses of sequence from virus obtained very early after infection, little divergence was seen from the founder viruses that initiated infection in each monkey . The sequence divergence for all week 2 semen isolates were not significantly different in control and vaccinated monkeys, with a mean distance among control animals of 0.27% and 0.15% among vaccinated monkeys (P = 0.4). These differences are consistent with the lower levels of viral replication in the vaccinated group SIV h monkey Table 1.env sequences from control animals showed a trend towards greater divergence (0.45%) than env sequences derived from the blood plasma of vaccinees . In week 16 sequences obtained from seminal plasma, the mean pair wise distance per animal was 0.31% and 0.22% for controls and vaccinees, respectively (P = 0.2). We next analyzed these sequence data using available algorithms to determine if changes in N-linked Env glycosylation sites had occurred during the sampling period We next analyzed blood and semen sequences from specimens obtained at week 16 following infection. Post set point, we were able to obtain sufficient numbers of concurrent semen- and blood-derived SIV sequences from 7 monkeys to allow a phylogenetic analysis of these viruses. Consistent with the lower level of virus replication in the vaccinated group, we observed reduced diversification of sequences obtained from the vaccinated monkeys, particularly in the seminal plasma. Specifically, blood-derived env by fixed-effects likelihood (FEL) We then analyzed the sequences from the inoculum and week 16 samples for evidence of selection on SIV Table S1). Among the sequences from inoculum and week 16 sequences, we found 12 codons in 6 monkeys (3 vaccinees and 3 from the control group) under positive selection. One of these codons was under positive selection in 2 different animals (codon 427 in AX93 and AY47).The presence of negative selection could not be determined as intra-host viral populations had not diversified sufficiently for such events to occur at detectable levels. The positive selection tests that reached significance (p<0.05) are indicated .We then conducted additional signature analyses using only compartmentalized animals, but a sample size of 4 compartmentalized animals would lack sufficient statistical power to identify robust predictive signatures of compartmentalization. Therefore, we adapted a related strategy used by Pillai et al. Reviewing the homologous signature sites in the SIV env sequences, we found 3 of 4 to be located 1–2 residues downstream of glycosylation sites. However, mutations in these sites (and in the neighboring 4 residues) were equally common in sequences derived from blood and semen, regardless of whether we included only sequences from compartmentalized animals. Thus, we were unable to confirm semen signatures in these SIV data.env sequences, we applied both phylogenetic analyses and the Slatkin-Maddison (SM) test from the HYPHY software package (v0.99b) env sequences were observed in the control and vaccinated animals. Importantly, we observed the mixing of env taxa from blood- and seminal plasma-derived virus, and the association of these sequences into closely related phylograms  . Therefore, there was weak evidence for virus compartmentalization in only 1 of the 14 monkeys by 2 weeks post-infection.While we did not observe topological evidence for virus compartmentalization between the semen and blood during early infection in any of the 14 monkeys analyzed, compartmentalization effects were also assessed using the SM test. Applying this metric, we observed no evidence of virus sequestration in week 2 blood- or semen-derived sequences in 13 of the 14 monkeys . Control<0.0001) . However)env sequences from seminal plasma and semen-associated cells from monkey CK7J using NJ- tree parameters .We also conducted an analysis, as described above, to ensure that isogenic sequences were not confounding the statistical analyses. This modified analysis did not significantly alter the findings as determined by tree topology. We also applied the SM test to phylogenies obtained from excluding duplicate sequences  and founenv, (data not shown). Next, we analyzed our data using a “segmented” SM-test based on env recombination breakpoints (not shown) and found specific regions of the SIV envelope from RMs C179 and AX89 were in fact significantly compartmentalized  among immediate descendents of that node. When these migration points were mapped directly onto phylogenies, we observed a concordance of semen compartmentalization with the number of migration events between these anatomic compartments, in individual monkeys. For example, the frequent migration events between the blood and male genital tract, as shown for monkeys C171, and DA2D  of inter-compartment gene flow would then account for the localized diversification of virus variants in the genital tissues.env diversification in the genital tract than in the blood of these monkeys. This finding is consistent with the lower level of SIV replication in the genital tract than in the blood at 16 weeks following infection. Moreover, the difference in virus diversification in these two anatomic compartments was particularly evident in the vaccinated monkeys after challenge , consistent with the low-level virus replication in the male genital tract after set point In fact, we observed less Although the male genital tract is generally considered to be an immune privileged compartment, inflammatory conditions present during acute SIV infection may facilitate the infiltration of both free virus and infected cells. Over time, differences between the blood and the genital tract, as a result of restrictions in gene flow would foster local replication in the genital tract leading to the compartmentalization observed at set point. Inflammation is a likely cause for the trafficking of cells from the peripheral circulation into the genital compartment, and may explain the observations of intermittent viral shedding in the semen of chronically HIV-infected men as a result of bacterial or viral infection of the male genital tract These apparent differences in selective pressure between the blood and male genital tract should also be considered in light of the reduced effective SIV population size in the genital tract as virus set point is achieved. Virus populations of reduced size and altered genotype relative to the blood may contribute to the described genetic bottleneck associated with HIV-1 transmission, highlighted by several investigators All Indian-origin rhesus monkeys used in this study and analysis were maintained according to the guidelines of the NIH Guide to the Care and Use of Laboratory Animals and the approval of the Institutional Animal Care and Use Committee of Harvard Medical School and the National Institute of Health. All monkeys were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All procedures were conducted in strict accordance with the Guide for Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard University.Macaca mullata) that were 4–6 years of age. All animals were confirmed as Mamu-A*01 negative by both PCR and MHC-amplicon sequencing. All animals were screened and found to be negative for simian immunodeficiency virus (SIV) and simian retrovirus type D (SRV-1) prior to the initiation of this study. All experiments were conducted in accordance with IACUC standards. Prior to, and routinely after SIV infection, all animals were monitored by physical exam, CBC with differential, and T cell subset analysis. The monkeys were housed under Biosafety level-2+ conditions.This study monitored 26 adult Mamu-A*01 negative male rhesus monkeys , directly into the saphenous vein.Rhesus monkeys were immunized using a heterologous prime/boost regimen that included both plasmid DNA and replication-defective recombinant adenovirus serotype 5 immunogens. One group of 13 monkeys received these vaccine vectors expressing SIVmac239 RNAlater . Samples were immediately frozen and maintained at −80°C until use.Viral levels were monitored in the semen and blood for 16 weeks after infection. Specimens were collected weekly for the first 6 weeks; then bi-weekly for the study duration. Semen was collected by eletroejaculation via rectal probe electrostimulation as, described Viral RNA levels were monitored in the seminal plasma and semen-associated cell fraction for 16 weeks after infection. Viral RNA was routinely isolated from 200 µL of cell-free, clarified peripheral blood or seminal plasma using the NucliSENS Isolation Kit  following the manufacturer's protocol.The SIV RNA standard was transcribed from the pSP72 vector containing the first 731 bp of the SIVmac239-Gag gene using the Megascript T7 kit (Ambion Inc.). RNA was isolated by phenol-chloroform purification followed by ethanol precipitation. All purified RNA preparations were quantified by optical density. RNA quality was determined by Agilent bioanalyzer RNA chip .gag RNA standard using the gene-specific primer sGag-R 5′CACTAGGTGTCTCTGCACTATCTGTTTTG-3′under the following conditions: the 50 µL reactions containing 1× buffer , 0.25 µM primer, 0.5 mM dNTPs (Roche), 5 mM dTT, 500 U Superscript III RT , 100 U RnaseOUT (Invitrogen), and 10 µL of sample. RT conditions were 1 hour at 50°C, 1 hour at 55°C and 15 minutes at 70°C. All samples were then treated with RNAse H (Stratagene) for 20 minutes at 37°C. All real-time PCR reactions used EZ RT PCR Core Reagents  following the manufacturer's suggested instructions under the following conditions: the 50µL reactions containing 1× buffer  glycerol, pH 8.2, .3 mM each of dATP, dCTP, dGTP, .6 mM dUTP, 3 mM Mn (OAc)2, .5 U uracil N-glycosylase, 5 U rTth DNA Polymerase, .4 uM of each primer, and 10µL of sample template. Following 2 minutes at 50°C, the polymerase was activated for 10 minutes at 95°C, and then cycling proceeded at 15 seconds at 95°C and 1 minute at 60°C for fifty cycles. Primer sequences were adapted from those described by Cline et al 5′-GTCTGCGTCATCTGGTGCATTC-3′, reverse primer s-Gag-R: 5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′, and the probe s-Gag-P: 5′-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-3′, linked to Fam and BHQ . All reactions were carried out on a 7300 ABI Real-Time PCR system (Applied Biosystems) in triplicate according to the manufacturer's protocols.QRT-PCR was conducted in a 2-step process. First, RNA was reverse transcribed in parallel with an SIV-Preliminary experiments were done to evaluate the quantitation of blood, and seminal plasma viral RNA levels and their correlation with known viral RNA quantities spiked into blood or semen sampled from SIV-uninfected monkeys (data not shown). To determine the linear range of the assay, RNA extraction efficiency and potential inhibition of reverse transcriptase activity, known quantities of serially diluted SIVmac251 viral stock were spiked into samples of peripheral blood, or semen that were recovered from three SIV-naïve monkeys. No significant differences in extraction efficiency were observed between the blood and samples derived from other compartments (data not shown). Furthermore, using the extraction methods described above, we observed no inhibition of RT activity or diminution of input RNA as a consequence of RNAse activity present in any of the virus-spiked specimens (data not shown). These findings indicated, therefore, that it was appropriate to employ this technique to isolate viral RNA from study samples of peripheral blood and semen from infected rhesus monkeys. The analytical sensitivity of our quantitative RT-PCR assay was determined as described gag DNA copies was calculated as described previously 5 semen-associated cells. All target sequences were normalized to Albumen levels in genomic DNA in multiplex reaction performed in duplicate .Cellular DNA was isolated using a QIAamp DNA Mini kit (QIAGEN). The number of SIV 2SO4, 0.2 µM primer OF6207 (5′ – GGGTAGTGGAGGTTCTGGAAG – 3′), 0.2 µM primer OR9608 (5′ – CTCATCTGATACATTTACGGGG – 3′), 0.025 units of Platinum Taq High Fidelity polymerase in a total volume of 20 µL. PCR mixtures were loaded into MicroAmp Optical 96-Well Reaction Plates (Applied Biosciences). PCR conditions were programmed as follows: 5 minutes at 94°C, 35 cycles of 15 seconds at 94°C, 30 seconds at 52°C, 4 minutes and 15 seconds at 68°C, followed by a final extension time of 10 minutes at 68°C 2SO4, 0.3 µM primer IF6428 (5′ – CGTGCTATAACACATGCTATTG – 3′), 0.3 µM primer IR9351 (5′ – CCCTACCAAGTCATCATCTTC – 3′), 0.025 units of Platinum Taq High Fidelity polymerase in a total volume of 20 µL. PCR conditions were: 5 minutes at 94°C, 45 cycles of 15 seconds at 94°C, 30 seconds at 51°C, 3 minutes and 30 seconds at 68°C, followed by a final extension time of 10 minutes at 68°C. Amplicons from cDNA dilutions resulting in less than 30% positive wells were sequenced at the Dana-Farber/Harvard Cancer Center DNA Resource Core.Viral cDNA was diluted in 96-well plates to yield fewer than 30% wells positive for amplification to ensure that positive amplifications were a result of a single cDNA . First-round PCR was carried out in a reaction mixture containing: 1× buffer , 0.2 mM dNTP mix, 2mM Mghttp://www.hiv.lanl.gov/content/sequence/GENE_CUTTER/cutter.html).Raw cDNA sequence data was assembled using GeneCodes Sequencher 4.8 DNA sequencing software. All assembled sequence contigs were manually corrected for individual ambiguous nucleotide errors and further quality controlled to exclude any amplicons derived from multiple templates according to that described by Learn et al, env divergence between animals receiving vaccine or sham-vaccinated controls. We modified the method described by Gilbert et al., to include a correction for small sample size bias , and restricted our analysis to animals those that had more than 3 sequences env through the first 17 nt of nef, to yield consistent phylogenetic signal among sequences, rather than variable signal due to 3′ gaps in some sequences but not others.The median and range of diversity was determined for each monkey, for both blood and semen, using a strategy suggested by Gilbert et al., that controls for the correlation of inter-sequence distances from the same animal and compared We used both Neighbor-Joining (NJ) and maximum likelihood (ML) for phylogenetic inference. The trees in env sequences, with a false-discovery rate correction applied to the resulting p-values to accommodate multiple tests.Signature detection utilized a phylogenetic correction to avoid over-counting amino acid substitutions among lineages of related sequences. To do this, we used methods described previously env sequences, we used the Slatkin-Maddison test s, the observed number of migration events required to yield the observed distribution of blood and semen sequences on a tree. It then assesses significance by comparing s with a re-sampled null distribution, which is obtained by holding the tree topology constant and randomly permuting the order of blood and semen labels across all leaves on the tree, computing s for each permutation. Statistical support for compartmentalization is attained when a large proportion of permutations yield more migration events than for the empirical s. That is, if fewer than 5% of 10,000 replicate permutations have at most s migration events, then P<0.05.To test for compartmentalization in contemporaneous blood- and semen-derived SIV Highlighter plots and confirmed by using the G.A.R.D. analysis, where significance was assessed at (P<0.001) .Analysis for recombination was performed by the visual inspection of Protein-coding regions for all Env were tested for specific codons under positive selection using FEL. A Neighbor-Joining tree and nucleotide substitution model were computed for each aligned gene before FEL analysis Statistical analysis was conducted using commercially available software GraphPad Prism version 4.0b . Linear regressions were conducted to determine the significance of correlations. Significance of differences between groups was determined using Kruskall–Wallis ANOVA as appropriate. Probability values of P<0.05 were interpreted as significant.Table S1Analysis of positively selected SIV env codons by fixed-effects likelihood test.(0.03 MB DOC)Click here for additional data file.Text S1Highlighter analyses of sequences from control monkeys AX89, C171, DA2D, CK7J, CM8X , and vaccinated monkeys D135, AX93, AY47, AY89 and C179 . Shown on each tree are 2 weeks after infection  or post set point at 16 weeks after SIV challenge  compared with inoculum sequences (black symbols). Brown points indicate sequences with three or more APOBEC-mediated G-to-A mutations compared with consensus sequences. Nucleotide polymorphisms in Highlighter plots are indicated as follows: A green, C cyan, G orange, T red, Other IUPAC code dark blue, Gap/no data grey.Viral sequence diversity in the semen and blood after i.v. SIVmac251 infection. NJ and (1.72 MB PDF)Click here for additional data file.


Chrysanthemum spp. However, the consequences of hybridization in this taxon have yet to be characterized.Hybridization is a major driver of evolution in plants. In a number of plant species, the process of hybridization has been revealed to be accompanied by wide-ranging genetic and epigenetic alterations, some of which have consequences on gene transcripts. The Asteraceae family includes a number of polyploid species, and wide crossing is seen as a viable means of genetically improving ornamental species such as C. nankingense × Tanacetum vulgare and C. crassum × Crossostephium chinense were employed to characterize, respectively, the genomic, epigenomic and transcriptomic changes induced by the hybridization event. The hybrids’ AFLP profiles included both the loss of specific parental fragments and the gain of fragments not present in either parent’s profile. About 10% of the paternal fragments were not inherited by the hybrid, while the corresponding rate for the maternal parent fragments was around 4–5%. The novel fragments detected may have arisen either due to heterozygosity in one or other parent, or as a result of a deletion event following the hybridization. Around one half of the cDNA-AFLP fragments were common to both parents, about 30% were specific to the female parent, and somewhat under 20% specific to the male parent; the remainder (2.9-4.7%) of the hybrids’ fragments were not present in either parent’s profile. The MSAP fingerprinting demonstrated that the hybridization event also reduced the amount of global cytosine methylation, since > 50% of the parental fragments were methylated, while the corresponding frequencies for the two hybrids were 48.5% and 50.4%.Amplified fragment length polymorphism (AFLP), methylation sensitive amplification polymorphism (MSAP) and cDNA-AFLP profiling of the two intergeneric hybrids Combining two different Asteraceae genomes via hybridization clearly induced a range of genomic and epigenomic alterations, some of which had an effect on the transcriptome. The rapid genomic and transcriptomic alterations induced by hybridization may accelerate the evolutionary process among progenies. Hybridization has contributed substantially to the evolution of higher plants, both in the context of extending genetic diversity and in enhancing adaptive speciation -3. At leAlthough been debated for more than a century, hybridization is considered to be a potent evolutionary force of genetic variation and functional novelty and occurs frequently in flower plant ,12. The In plant breeding and domestication process, hybridization is a powerful method to import excellent genes and exquisite traits into hybrids (either caused by additive or non-additive effects), which results in the phenotypic superiority of a hybrid over its parents with respect to traits such as greater biomass, speed of development and yield ,27. CompC. nankingense) to decaploid (eg. C. crassum) which is generally considered to be an advanced subjects and at the forefront of the evolution [Chrysanthemum even in Asteraceae [C. nankingense × Tanacetum vulgare and C. crassum × Crossostephium chinense hybrids.Asteraceae is a large group of angiosperms distributed all over the world includes ploidy states ranging from diploid , E2 + M6, E3 + M2, E4 + M3, E4 + M8, E6 + M7, E7 + M3, E8 + M3 and E8 + M7; each primer included three selective bases  Long Ranger; 36 cm in length) for 2 h at 65 W. Only reproducible fragments in the size range 100–500 bp of two replications were recorded as present (1) or absent (0).The AFLP protocol used to profile the genomic DNA and cDNA was a minor modification of the one described by Vos et al. . The firHpaII  and MspI , in combination with EcoRI . Methylation polymorphisms at 5′-CCGG-3′ tetranucleotide sites generate differences between the EcoRI-HpaII (H lanes) and EcoRI-MspI (M lanes) profiles [EcoRI and 20 U HpaII or 10 U EcoRI and 10 U MspIat 37°C for 12 h. The digested fragments were ligated to 5 pmol EcoRI adaptor and 50 pmol HpaII–MspI adaptor by incubation with 4 U T4 DNA polymerase  at 16°C for 4 h. Amplicons derived from a pre-selective amplification based on EcoRI pre-selective primer and HpaII/MspI pre-selective primer formed the template for a subsequent selective amplification.Selective amplification reaction was based on one of nine primer combinations  were present in one or both of the parents. The same frequency applied for the C. crassum × Cr. chinense hybrid  and epigenetic adjustment [Cucumis[Brassica[Tragopogon[C. nankingense × T. vulgare and the C. crassum × Cr. chinense combinations. The deletion events were likely to have occurred very early in the process of hybrid zygote formation.A variety of analytical platforms has been exploited to show that justment ,17,46. Rjustment ,47-50. Ijustment , and an  [Cucumis, Brassic[Brassica and Tragragopogon. ExtensiC. nankingense, and respectively 275, 164 and 133 in T. vulgare; the equivalent numbers of fragments in C. crassum were 261, 171 and 153, and in Cr. chinense 268, 162 and 142  × T. vulgare  and 50.4% for C. crassum (55.4%) × Cr. chinense . With respect to the hemi-methylated sites, the respective U values were only 0.21 and 0.47.Meanwhile, polymorphic fragments were also scored as methylation changes between hybrids and parents. The amount of cytosine methylation in the hybrids were 48.5% for 1 hybrid.Present results suggested that the adjustments of DNA methylation patterns occurred widely at various genomic sites in each of the hybrid plants Table . CombiniC. nankingense × T. vulgare cross and 52.2% in the other cross; (b) fragments which were specific to the female parent and which were inherited by the hybrid (29.6% and 28.6%); (c) fragments which were specific to the male parent and which were inherited by the hybrid (16.9% and 14.5%), and (d) fragments in the hybrid which were not present in either parent (3.0% and 4.7%; Figure C. nankingense, 22 of T. vulgare and 9 of C. crassum, 19 of Cr. chinense cDNA fragments missed in cDNA-AFLP detection (Table Both hybrids grow larger and faster than the best of their parents Figure D, F 36,,36,37. cTranscriptomic studies of hybridization in plants have revealed that patterns of gene transcripts likely have a profound effect in a hybrid context . In spitC. nankingense × T. vulgare and C. crassum × Cr. chinense. The forced union of two distinct genomes induced many changes to both the genome and the transcriptome. The former changes were largely brought about by the elimination of DNA, while the latter reflected in addition the effect of altered amount of cytosine methylation. Together, these rapid changes could drive the evolutionary process of the freshly formed intergeneric hybrids.In conclusion, large scale genomic, epigenomic and transcriptomic changes accompanied the process of hybridization in the crosses The authors have no competing interests to declare.Conceived and designed the experiments: HW FC JJ SC. Performed the experiments: HW FC JJ. Analyzed the data: HW JW. Contributed reagents/materials/analysis tools: WF ZG. Wrote the paper: HW JJ SC. All authors read and approved the final manuscript.


United States pediatric guidelines recommend that childhood obesity counseling be conducted in the primary care setting. Primary care-based interventions can be effective in improving health behaviors, but also costly. The purpose of this study was to evaluate the cost of a primary care-based obesity prevention intervention targeting children between the ages of two and six years who are at elevated risk for obesity, measured against usual care.High Five for Kids was a cluster-randomized controlled clinical trial that aimed to modify children’s nutrition and TV viewing habits through a motivational interviewing intervention. We assessed visit-related costs from a societal perspective, including provider-incurred direct medical costs, provider-incurred equipment costs, parent time costs and parent out-of-pocket costs, in 2011 dollars for the intervention (n = 253) and usual care (n = 192) groups. We conducted a net cost analysis using both societal and health plan costing perspectives and conducted one-way sensitivity and uncertainty analyses on results.The total costs for the intervention group and usual care groups in the first year of the intervention were $65,643  and $12,192 . The mean costs for the intervention and usual care groups were $259  and $63  per child, respectively, for a incremental difference of $196  per child. Children in the intervention group attended a mean of 2.4 of a possible 4 in-person visits and received 0.45 of a possible 2 counseling phone calls. Provider-incurred costs were the primary driver of cost estimates in sensitivity analyses.High Five for Kids was a resource-intensive intervention. Further studies are needed to assess the cost-effectiveness of the intervention relative to other pediatric obesity interventions.NCT00377767.ClinicalTrials.gov Identifier:  In response to the growing prevalence of childhood obesity, the 2010 Patient Protection and Affordable Care Act promotes the utilization of preventive care, mandating that insurers cover obesity screening and counseling services as part of insurance policies ,4, but iOnly a dozen analyses in the peer-reviewed literature detail the costs of delivering childhood obesity interventions -16. JustHigh Five for Kids was an obesity prevention program developed for 2-6.9 year old children who were obese, or overweight with at least one overweight parent . The intHigh Five for Kids was a cluster-randomized controlled trial conducted from 2006-2008 in 10 non-profit private pediatric offices of Harvard Vanguard Medical Associates, a multi-site group practice in eastern Massachusetts. There were 5 intervention groups (n = 253 children) and 5 usual care practice groups (n = 192 children). The intervention was based on the Chronic Care Model, which suggests that all members of the pediatric care practice team should be actively involved in an intervention in order to successfully change patient behaviors and improve health outcomes . As suchWe assessed costs of the motivational interviewing intervention, including the cost of study-related visits incurred by the provider, costs incurred by the parents of children while attending study visits, intervention training costs, and the cost of materials and equipment for the intervention and usual care groups. The inclusion of costs incurred by the parents of children in the trial allowed us to conduct our analysis from the societal perspective, considering the investment of time and financial resources made by every person involved in the intervention . Costs wProvider-incurred costs included the cost of study-related visits, materials, and equipment. We did not measure costs related to healthcare utilization of non-study-related visits. In the base case, we calculated the cost of study-related visits and phone counseling using a micro-costing approach by multiplying APC wage data by the time APCs spent counseling parents in person and by phone. We derived overhead costs using published estimates of overhead costs per visit in a pediatric practice  and dataWe administered a survey at the end of year one of the study to estimate parent non-medical and time costs. Parents reported their time, time required for childcare for other children, transportation, and out-of-pocket costs associated with study visits. Parent time costs were valued using the 2007 mean adult US wage rate ,23. ChilUsing a percentile method approach, we conducted uncertainty analyses to assess variability in results from potential sampling bias . We usedSensitivity analyses on alternative costing approaches were conducted to test the robustness of results. First, we considered an approach that included training costs in total intervention costs. Training costs are not conventionally considered in base case analyses, rather, they are typically treated as fixed costs. Secondly, we considered an approach that used health plan reimbursement data to calculate the costs of study visits from a payer perspective as opposed to the micro-costing approach used in the base case. Lastly, we considered a costing approach that annuitized costs for durable equipment that could be used by practices after the completion of the study.We calculated training costs for intervention site clinical staff and APCs. As the key intervening clinicians, APCs participated in eight 1 to 3 hour-long on-site training sessions conducted by experienced APCs. Intervention site clinical staff  participated in one 1-hour training session, and medical assistants in both study arms attended an additional 1-hour session to learn how to use scales and stadiometers. We obtained wage data for APCs from primary study data. Intervention site clinical staff hourly wages were estimated from the 2007 Bureau of Labor Statistics survey .As an alternative to the micro-costing approach employed in the base case, we used reimbursement data obtained from the health plan to calculate the cost of study visits from the health plan perspective. While all intervention participants attended study visits for counseling, reimbursement data were available for only 238 out of the estimated 504 visits because participants did not submit invoices for reimbursement of visit co-pays. We assumed visit cost data was missing at random. We imputed missing visit costs as the mean non-missing visit cost of all children in the trial using data on the number of study visits each child attended.Our base case analysis took a conservative approach to estimating equipment costs, allocating the entire costs of scales and stadiometers to the intervention. However, these equipment costs could be considered capital equipment, with a life expectancy beyond the two-year study time period. In a sensitivity analysis, we considered a more liberal costing approach in which costs were annuitized over the useful life of the equipment. We assumed that scales and stadiometers could both be used for 10 years before being replaced.The total costs for the intervention group were $65,643  or $259  per child. The total costs for the usual care group were $12,192 , or $63  per child  and $8,025 in usual care ($42 per child). Expected intervention group health plan costs, estimated using imputation, were $162,255 total, or $641 per child. Costs estimated from the health plan perspective yielded cost estimates that were approximately 10% to 147% higher than base case results calculated using the societal costing perspective Table .In sensitivity analyses that considered two-year annuitized costs as opposed to the full cost of scales and stadiometers decreased costs in the intervention and usual care groups. Usual care costs decreased 19% from $63 to $51 per child. Intervention costs decreased only 3% from $259 to $251 per child . Provider-incurred costs were the primary driver of intervention costs. The cost of the intervention was higher in sensitivity analyses that considered the inclusion of training costs and costs calculated from the perspective of the health plan.2) and BMI z-score [This analysis of the cost of High Five for Kids quantifies the economic burden of one primary care-based motivational interviewing intervention. Data on training costs and direct medical costs may be of particular interest to providers seeking to implement such an intervention, and information on costs from the perspective of the health plan may be of particular interest to payers. In order to get a better sense of the value of the intervention—the gains in health relative to the dollars spent on care—a cost-effectiveness analysis should be conducted. High Five for Kids measured program effectiveness in terms of changes in body mass index  intervention, the only other primary care-based child obesity prevention program for which costs have been evaluated, were more costly than High Five for Kids. The net cost of delivering the LEAP intervention was approximately $852 (LEAP Phase 1) and $1,237 (LEAP Phase 2) per child from the health plan cost perspective. These estimates of the cost of LEAP include any reported costs of study-related visits with the primary care practitioner and equipment costs, but exclude any family-incurred time or out of pocket expenses -11,29. UWhen making comparisons to other cost analyses, it is important to consider intervention characteristics or assumptions made in analyses that can affect per capita cost estimates. It is difficult to compare health plan perspective costs from other countries because the health services and unit costs may not be comparable . MoreoveOur use of a micro-costing approach to calculate provider-incurred costs provides estimates of the marginal cost of the intervention per child treated, which may be useful to others planning programs. The micro-costing approach estimated substantially lower intervention costs than those generated using health plan perspective costs because health plan perspective costs may represent prices that could vary between practices and insurers, and may incorporate substantial administrative costs . The micWe excluded training costs from our primary analysis, consistent with the conventional framework for economic evaluation that these were start-up fixed costs that would not vary materially with the scale of the intervention . HoweverBecause we were missing some health plan costs, we relied on imputation to derive expected costs from the health plan perspective. But because the children had a limited number of insurance providers and saw providers in one network, we believe that the imputed costs are fairly representative of actual costs. Secondly, the study was focused on a small number of practices in eastern Massachusetts, which may lead to concerns about the generalizability of study results to other practices in the U.S. However, the results found using the micro-costing approach represent national costs, improving generalizability of the findings. Lastly, the visit compliance among intervention subjects in primary care-based interventions such as High Five for Kids and LEAP was lower than expected and some usual care patients unexpectedly received weight counseling visits . As the The net cost of the primary care-based High Five for Kids motivational interviewing intervention was $196 per child. In order to help decision makers decide whether to adopt such a program, future research should measure the value of High Five for Kids relative to other interventions using a cost-effectiveness analysis. A decision-analytic simulation model could explore scenarios in which the intervention would have been more or less effective in addition to illustrating the impact of the persistence of intervention effects beyond the trial. Estimating the short- and long-term value of the intervention using simulation models is also an important next step in appraising interventions.CI: Confidence interval; BMI: Body mass index; APC: Advanced practice clinicians; LEAP: Live eat, and play intervention.The authors declare that they have no competing interests.EMT, MWG, SLG, LAP, and DRW were responsible for the study concept and design. EMT, MWG, KHH, and CMH were responsible for the acquisition of data. DRW was responsible for the analysis and interpretation of data and drafted the manuscript. All authors reviewed the manuscript for intellectual content and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/14/44/prepub


Apoptosis is common to all multicellular organisms. Apoptosis can be triggered by the extrinsic (death receptor (DR)) or the intrinsic  death pathways. CD95 (APO-1/Fas) is a prototypic member of the DR family. This review is focused on the mechanisms of CD95 (APO-1/Fas)-mediated apoptosis and the role in the apoptosis of the death effector domain (DED)-containing proteins: pro-apoptotic protein procaspase-8 and anti-apoptotic protein c-FLIP. Gaining insights into these processes will improve our understanding of the pathogenesis of diseases such as cancer, autoimmunity and AIDS, and will open new approaches to rational treatment strategies. CD95  is a member of the death receptor (DR) family, a subfamily of the tumor necrosis factor receptor superfamily ; 3. DD aCrosslinking of CD95 with its natural ligand, CD95L CD178) , or with8 , or wiDED proteins at the DISC play a central role in the regulation of DR-induced apoptosis. Recruitment of procaspase-8 to the DISC, followed by its activation at the DISC and formation of active caspase-8 heterotetramers, triggers the apoptotic pathway. Recruitment of c-FLIP proteins to the DISC has the opposite effect: c-FLIP proteins block procaspase-8 activation at the DISC and thereby apoptosis induction. Therefore, life/death decisions at the DISC are defined by the balance of two DED proteins: procaspase-8 and c-FLIP. In this review we describe in detail the DED-proteins procaspase-8 and c-FLIP and the mechanism of their pro- and anti-apoptotic action.Procaspase-8  belongs to the family of caspases ; 10. CasAll caspases are produced in cells as catalytically inactive zymogens and must undergo proteolytic processing and activation during apoptosis . The effProcaspase-8 is activated at the DISC . Two isoThe activation of procaspase-8 is believed to follow an 'induced proximity' model in which high local concentrations and favourable mutual orientation of procaspase-8 molecules at the DISC lead to their autoproteolytic activation . There ic-FLIP, also known as FLAME-1/I-FLICE/CASPER/CASH/MRIT/CLARP/Usurpin, is a well-described inhibitor of DR-mediated apoptosis. The current view on c-FLIP proteins is shown in In addition, two cleavage products of c-FLIP have been reported until now: p43-FLIP and p22-FLIP ; 24. P43DED proteins procaspase-8 and c-FLIP play a central role in the regulation of DR-induced apoptosis and might also induce the NF-κB pathway . RegulatInterestingly, in the cytosol, interactions between procaspase-8 and c-FLIP have been reported to induce the NF-κB pathway rather than apoptotic pathways. Recently, a new NF-κB-activating pathway initiated by procaspase-8 has been described. It has been shown that, independently of DR stimulation, non-apoptotic procaspase-8 activity generates the p22-FLIP cleavage product that leads to the induction of NF-κB The balance between DED-containing proteins may provide sensitive signaling check points that cells use for signaling cross-talk and switching between apoptosis-resistant and sensitive phenotypes and, thus, between life and death. Furthermore, the balance between DED-proteins also depends on the subcellular localization. The regulation by c-FLIP and procaspase-8 of life/death decisions at the DISC is different from the cytosolic events. At the DISC, c-FLIP mostly acts as a devoted procaspase-8 inhibitor, while in the cytosol it uses procaspase-8 activity to initiate cleavage to p22-FLIP and the subsequent NF-κB induction


Chordopoxvirinae subfamily of the Poxviridae family. APVs are distributed worldwide and cause disease in domestic, pet and wild birds of many species. APVs are transmitted by aerosols and biting insects, particularly mosquitoes and arthropods and are usually named after the bird species from which they were originally isolated. The virus species Fowlpox virus (FWPV) causes disease in poultry and associated mortality is usually low, but in flocks under stress  mortality can reach up to 50%. APVs are also major players in viral vaccine vector development for diseases in human and veterinary medicine. Abortive infection in mammalian cells (no production of progeny viruses) and their ability to accommodate multiple gene inserts are some of the characteristics that make APVs promising vaccine vectors. Although abortive infection in mammalian cells conceivably represents a major vaccine bio-safety advantage, molecular mechanisms restricting APVs to certain hosts are not yet fully understood. This review summarizes the current knowledge relating to APVs, including classification, morphogenesis, host-virus interactions, diagnostics and disease, and also highlights the use of APVs as recombinant vaccine vectors.Avipoxviruses (APVs) belong to the  Chordopoxvirinae subfamily of the Poxviridae family [Avipoxviruses (APVs) are among the largest and most complex viruses known. APVs belong to the e family . They ine family -4. APVs e family . Althouge family , our knoe family ,8. Curree family ; Table 1e family ,9. APV-ve family -14, and Parus major), have failed to multiply on CAM of chicken embryos [Avipoxviruses are large, oval-shaped enveloped viruses whose genome consists of double stranded DNA ranging in size from 260 to 365 kb . Unlike  embryos . APVs ar embryos ,5. Histo embryos ,18.Great discoveries made in the mid-nineteenth century facilitated major advances in pox virology. Based on the report by Bollinger  on poxviAvipoxvirus belong to the subfamily Chordopoxvirinae which shares several biological features with other poxviruses [Members of the genus xviruses ,8. Currexviruses , avian pxviruses . These sAvipoxviruses share several morphological, biochemical and physiochemical features with other poxviruses. Virus particles measure 270 × 350 nm and are composed of an electron dense, centrally located core and two lateral bodies that are visible in fixed and stained ultra-thin sections. In negative stained preparations, such as phosphotungstic acid (PTA) the membrane displays an outer-coat composed of a random arrangement of tubules ; Figure Avipoxviruses have low G+C content (30 to 40%) and consist of a single linear molecule of double-stranded DNA of between 260-365 kb. The central region of the genome is flanked by two identical inverted terminal repeats (ITRs) which are covalently linked by hairpin loops and contains several hundred closely spaced open reading frames . The cenComplete genomes of the two most studied APVs, FWPV US , FWPV-FP9 (a plaque-purified tissue culture-adapted attenuated European virus) and a CNPV virulent strain  have been sequenced ,8,27. Alin vitro characterization of APVs [Virulence genes are generally of non-conserved nature and influence the pathological profile of viruses in an infected host. These genes are important in viral evolution and have been used in studies to provide insight into how some poxviruses evolve strategies to ensure their replication . Many of of APVs ,8,38,39, of APVs . MoleculCompared to other poxviruses, such as vaccinia virus, mechanisms that account for APV pathogenesis is poorly understood. APVs have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into host cells. Like many other DNA viruses, APV probably devotes much of its genes to allow it to evade host immune responses. Such viral genes commonly encode proteins that are critical for the virus to undergo molecular transformation that leads to successful membrane fusion, penetration and intracellular transport. These includes genes that encodes proteins which act on early innate pathways such as pathways involving interferon , patternSince the first isolation of APVs in cell culture, these viruses have been recognized as highly host specific. They are believed to replicate only in avian cells, notably chicken embryo fibroblasts  . CEF celThe cellular entry and exit of APV is complicated by the existence of at least two distinct forms of virus that can productively infect cells, namely the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). These two forms are surrounded by different lipid membranes and surface proteins that are yet to be fully characterized.After virus binding to cellular membranes, a fusion step, which is generally poorly understood, results in the release of the virion core into the cytoplasm of the cell . The relIn permissive cells, the first viral structures detectable by electron microscopy are the crescent-shaped forms Figure , consistPoxvirus tropism may not be dependent upon specific cell surface receptors, but rather upon the ability of a given cell to provide intracellular complementing factors needed for productive virus replication, and on the ability of the specific virus to successfully manipulate intracellular signaling networks that regulate cellular antiviral processes following virus entry . APVs haBranta sandvicensis) and the Palila (Loxioides bailleui), were compared with FWPV in specific-pathogen-free chickens. Immune responses were measured by ELISA before and after immunization with Hawaiian APVs and after challenge with FWPV. Both isolates from Hawaiian birds developed only a localized lesion of short duration at the site of inoculation in chickens and did not provide protection against subsequent challenge with virulent FWPV, in which severe lesions were observed. In contrast to high antibody response in chickens immunized with FWPV, birds immunized with either of the two Hawaiian isolates developed low to moderate antibody responses against viral antigens [Branta canadensis) was transmissible to domestic goose, but not to chickens or domestic ducks [Pica pica) and great tits (Parus major) did not infect young chickens [(Gymnorhina tibicen) produced lesions in chickens. These studies were based on clinical manifestations in the chickens and suggest host specificity and pathogenicity.APV infections are associated with significant levels of morbidity and mortality in domestic and wild bird populations ,60. Mostantigens . Pathogeantigens , turkeysantigens ,63,64. Aic ducks . Pigeonpic ducks . Poxviruchickens , howeverDespite the worldwide prevalence of APV infections, experimental infection studies in birds using APVs have centred on relatively few viral isolates. Analyses of variation have essentially focused on a FWPV strain termed the prototype, while a minority of experimental studies have been reported on CNPV, quailpox, juncopox, and pigeonpox virus isolates ,48,49. IBamHI, EcoRI, and HindIII endonucleases and distinct fragment patterns were observed between the isolates. The patterns of three quailpox virus isolates were similar to each other with a high proportion of co-migrating fragments. However, when immunogenic proteins of three FWPVs, two quailpox viruses, a juncopox virus, and a pigeonpox virus isolates were examined by immunoblotting, shared as well as unique antigens were detected. The greatest disparity was observed between quailpox virus and FWPV [Our present understanding of the antigenic variation of APVs has been based on a limited number of virus isolates in assays that includes complement-fixation, passive hemagglutination, agar-gel precipitation, immunoperoxidase, virus neutralisation and immunofluorescence ,49,65. Iand FWPV ,49, indiand FWPV . These aand FWPV .Spheniscus demersus), was most closely related to turkeypox virus, ostrichpox virus and pigeonpox virus [Understanding the phylogenetics of APVs is essential to the understanding of host specificity and virulence, but also to provide insights into the variation of different viruses. Although the complete genome sequences of FWPV and CNPV are available ,8, littlox virus .During avipox outbreaks, mortality can reach 80 to 100% in canaries and other finches. This is in contrast to a generally lower mortality seen in chicken and turkey . TransmiClinical features of infected birds show multiple skin lesions varying from papules to nodules. Gross lesions in both the cutaneous and the diphtheritic forms, seen on birds and during necropsy, are usually sufficient to suspect APV infection . HoweverSuspicion of clinical signs of APV infection can if possible be supported by necropsy, especially if the oral cavities to reveal the diphtheritic form. Further, histopathology on tissue sections using the classic Wright's Giemsa stain may reveal typical large, solid or ring-like, eosinophilic intracytoplasmic inclusions known as Bollinger bodies ; Figure Demonstration of infectious virus by inoculation of homogenates of clinical samples of typical APV skin lesions onto the CAM of embryonated hen's eggs is the gold standard method for diagnosis of APV, although some strains of APV do not grow readily on chicken embryos . Eggs arAPV are increasingly being detected and characterized by PCR, Restriction fragment length polymorphism (RFLP), Southern blot hybridization, and cycle sequencing, directed at specific genes such as the 4b core protein gene ,23. PCR The conventional serological techniques of passive neutralization and agar-gel immunodiffusion are in continued global use for surveillance and disease control efforts in domestic poultry species ,76, despThe challenges of controlling APV disease in poultry are driven by economics, and require strategies that keep cost low while maintaining treatment efficacy. Prophylaxis can be achieved by vaccination . Doyle  reportedIn the poultry industry, prophylactic measures against FWPV are achieved primarily by vaccination with live FWPV or antigenically similar pigeonpox virus strains produced in CEF cells . In the + T cell-dominant response, whereas modified vaccinia virus Ankara (MVA) induces a stronger CD8+ T cell response with accompanying CD4+ T cell responses that are required for protection [In 1796 Edward Jenner  publisheotection . AlthougCurrently, representatives of a wide range of virus families are under intensive development as vaccine vectors for human or veterinary use. Of these, FWPV and CNPV appear to be of great interest as vectors, and some veterinary APV-vectored vaccines are already licensed and in commercial use in North America, South America and Europe Table . The mosAPV-vectored vaccines have been used as vaccines against several animal infections including West Nile virus (WNV), canine distemper virus, feline leukemia virus, rabies virus, and equine influenza virus ; Table 2in vivo in a manner similar to their wild-type parental viruses. Another reason may be fear of risk of spontaneous recombination between virus vectors and naturally occurring viral relatives in the ecosystems in which the vaccine is used. Even if APVs are not generally expected to replicate in mammals, the vaccine vectors may reach bird populations via animal populations. It is also possible that the vector, through spontaneous recombination and mutation events, may restore its replication competence. To cater for this, the aim during design and development of a virus vector is always to introduce at least two gene deletions crucial for viral to undergo a full replication cycle to assure a very low probability that replication competence could be restored. To our knowledge, such reversions have not been identified in clinical trials of APV-vectored vaccines. In addition to concerns regarding reversion or recombination, another safety signal was recently identified in an in vitro experiment that showed APV replication in cell clones derived from embryonic bovine trachea [Despite these notable advances in APV-vectored vaccine development, the list of licensed viral vectored vaccines for human medicine is short, with only a few vaccines that have entered clinical trials ; Table 3 trachea  and Syri trachea . These fAPVs cause disease of economical importance for the poultry industry, and also in pet and wild birds. Thus, prophylactic measures, such as vaccination, will always be required, and there is a need for more efficient and safe vaccines. One promising approach is the use of APVs as vectors for recombinant vaccines, increasing the efficacy and avoiding the potential contamination with REV and other agents. Many recombinant APV constructs are already licensed for use in veterinary medicine, and a range of vaccine candidates are currently being tested for use in vaccines against numerous infectious diseases in animals and man. Thus, it is likely that recombinant APV-vectored vaccines in the near future will also be used against human diseases. APVs have many advantages as vaccine vectors, including a large genome which allows for the inclusion of many heterologous genes, such as genes coding for antigens, cytokines and other immuno-modulating factors. The major safety argument for using APVs rather than vaccinia virus or other mammalian viruses as vectors, is that APVs are not zoonotic and are not able to conduct a full replication cycle in mammals. However, it was recently shown that FWPV was able to replicate and produce progeny virions in some established mammalian cell lines. This illustrates the fact that general knowledge of APVs is scarce. Indeed, only a few isolates have been characterized and classified. New molecular tools have led to a greater resolution of factors and mechanisms that restrict viruses to certain hosts, for example HIV and SARS. Mechanisms of host restriction, pathogenicity, host immunity and viral immune evasion strategies are of crucial importance regarding use of APVs as vectors in multispecies-targeted vaccines. A good understanding of the molecular properties of APVs underpins the development of safe APV-vectored vaccines.ALVAC: attenuated canarypox virus CNPV; APVs: Avipoxviruses; ATCC: American Type Culture Collection; BHK: Baby hamster kidney; CAM: chorioallantoic membrane; CEA: carcinoembryonic antigen; CEF: chicken embryo fibroblast; CHO: Chinese hamster ovary; CPE: cytopathic effect; CSP: circumsporozoite protein; EEV: extracellular enveloped virus; ELISA: enzyme linked immunosorbent assay; Env: envelope glycoprotein; F: fusion protein; FP9: attenuated fowlpox virus 9; FWPV: fowlpox virus; G: glycoprotein G; Gag: group specific antigen; gp120: glycoprotein 120; gp160: glycoprotein 160; HA: hemagglutinin; HA5: Hemagglutinin from influenza virus H5; HIV-1: human immunodeficiency virus 1; HN: haemagglutinin-neuraminidase protein; ICAM1: intercellular adhesion molecule 1; IEV: intracellular enveloped virus; IMV: intracellular mature virus; ITRs: inverted terminal repeats; IV: immature virus; LFA3: lymphocyte function-associated antigen 3; LIPO-5: Lipopeptide 5; MDV: Marek's disease virus; MOI: multiplication of infection; NIH: US National Institutes of Health; PCR: polymerase chain reaction; prM-E: pre-membrane and envelope proteins; Pro: protease; PTA: phosphotungstic acid; REV: Reticuloendotheliosis virus; RFLP: restriction fragment length polymorphism; TEM: transmission electron microscopy; TLR: Toll-like receptor; TRICOM: triad of costimulatory molecules; VACV: vaccinia virus; WNV: West Nile virus;The authors declare that they have no competing interests.SCW and MT contributed equally in drafting and reviewing the manuscript. All authors have read and approved the final manuscript.