Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)—and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species—Eurasian wigeon ( These outbreaks were associated with the first human infections caused by the HPAI H5N1 virus and with the spillover of HPAI H5N1 virus to wild birds. The hemagglutinin (H) gene of the HPAI H5N1 virus diversified into multiple genetic lineages (“clades”); more recently, reassortment between the HPAI H5N1 virus and the low pathogenic avian influenza (LPAI) viruses resulted in HPAI viruses with neuraminidase (N) genes  and other genes of LPAI virus origin5. From China, H5 GsGd virus has been introduced to other Asian countries, the Middle East, Africa, and Europe. Within Europe, HPAI H5N1 GsGd virus has been detected in multiple countries in 2004 (clade 1), 2005/2006/2007 (clades 2.2 and 2.2.1), and 2008/2009/2010 (clade 2.3.2)7. In November and December of 2014, the HPAI H5N8 GsGd virus 8 was detected in wild birds and poultry in various countries of Asia, Europe, and—for the first time—North America. The global spread of HPAI H5N8 virus in 2014/2015 raises the question whether this H5 virus was better adapted to wild birds  than the HPAI H5 virus from before 2014/2015.Avian influenza viruses—in particular highly pathogenic avian influenza (HPAI) viruses—form a continuous threat to the poultry industry, public health, and to some wild bird species. Since the emergence of the HPAI H5N1 virus in the poultry in China in 1996, H5 HPAI viruses that share a common ancestral virus (A/goose/Guangdong/1/96 [GsGd]) have continued to cause outbreaks in poultry9. Furthermore, experimental infection studies showed that ducks of different species infected with HPAI H5 GsGd virus may excrete virus without showing clinical signs. Despite HPAI H5 GsGd viruses being highly pathogenic to gallinaceous poultry, these viruses were not uniformly pathogenic to domestic ducks11 or wild ducks of different species 13. Also, HPAI H5 GsGd virus-specific antibodies have been detected in free-living wild ducks of several species in Asia and Europe suggesting that these birds survive HPAI H5 GsGd virus infection15. Nevertheless, most wild birds in which HPAI H5 GsGd viruses have been detected were moribund or dead, and so far, field observations did not clarify the variable effect of the HPAI H5 GsGd virus infection on individuals, host species, and populations. Thus, field observations as well as experimental studies provide evidence for migratory birds as long-distance vectors for some HPAI H5 GsGd viruses, but indicate high variation in pathogenicity of the HPAI H5 GsGd virus infection, depending on the virus clade, host species, and individual host.The long-distance spread of HPAI H5 GsGd virus from Asia to other continents is thought to occur via migratory birds or poultry trade. With respect to the role of wild birds, timing and geographical location of HPAI H5 GsGd virus detection in wild birds corresponded with timing and direction of wild bird migration from shared breeding grounds, where multiple major flyways diverge toward separate wintering grounds. For instance, the newly emerging HPAI H5N8 GsGd virus was detected along major flyways in Russia at the end of summer in 2014, followed in time by detection of wild birds and/or poultry in Europe and in North America at the end of fall in 2014. A large-scale genetic analysis of HPAI H5 GsGd viruses, combined with analyses of epidemiological and ornithological data, indicated that the main routes of long-distance geographical spread were most probably via the infected migratory birds16.In this study, we question whether the HPAI H5N8 virus clade 2.3.4.4 group A detected in Europe in 2014 has different virulence in different species of wild waterbirds than the HPAI H5N1 virus detected in Europe before 2014. We experimentally infected four species of wild ducks: Eurasian wigeon, common teal, mallard, and common pochard with an avian isolate of HPAI H5N8 virus from Europe obtained in 2014. We chose the ducks because they are abundant, migratory, and an important group in the epidemiology and the ecology of influenza A viruses in the wild. We chose these particular duck species because of their abundance, preference for freshwater habitats, geographical distribution, and migratory pattern spanning Asia, Europe, and Africa. Additionally, we chose these particular duck species as well as these methods of inoculation and analysis to enable a comparison with previous experimental infections of wild ducks with HPAI H5N1 virusAythya (common pochard) and three species of dabbling ducks belonging to the genus Anas . All four duck species became infected with HPAI H5N8 virus, 63% (20/32) of the ducks according to virus isolation and 100% (32/32) of the ducks according to RT-PCR  were inoculated experimentally: one species of diving duck belonging to the genus CR Table . In contP < 0.05) and RT-PCR   0–4 dpi, ) Tables , S2, S3.) Tables , S2.Fig.HPAI H5N8 virus that was excreted from the pharynx likely originated from the respiratory tract, based on virus isolation, immunohistochemistry (IHC), and RT-PCR. Infectious virus was isolated from the air sacs, trachea, and/or lung at 4 dpi. Specifically, infectious virus was isolated from the air sac , from trachea (common pochard), and lung  at 4 dpi Table . Based ot-test, P < 0.0003). According to virus isolation, pharyngeal excretion exceeded cloacal excretion for Eurasian wigeon only . However, virus titers were low for the other duck species , but did differ according to RT-PCR   and common pochard , but not compared to the common teal . The common teals excreted significantly more virus from the cloaca compared to mallard , but not compared to the common pochard . According to virus isolation, and similar to pharyngeal excretion, cloacal excretion varied substantially among individuals within the species  and limited to Eurasian wigeons (4/8) and common teals (3/8) Table . However, 22% and1) Table , S3. Bases Table , S2.TablHPAI H5N8 virus excreted from the cloaca may have originated from the kidney, pancreas, and/or liver, as infectious virus was isolated from these organs at 4 dpi , there was no evidence of HPAI H5N8 virus infection in the brain of any ducks according to IHC, histopathology, or virus isolation. Nonetheless, according to RT-PCR, weak positive values  were found in the brains of Eurasian wigeon (1 of 4), common pochard (1 of 4), and common teal (3 of 4) Table –S3.17. In contrast to the experimentally infected ducks, HPAI H5N8 virus was isolated from most tissues of experimentally or naturally infected chickens  and the diving duck species (common pochard) excreted HPAI H5N8 virus in the absence of debilitating disease and therefore may act as long-distance vectors for HPAI H5N8 virus  as detected in Europe in 2014. The absence of disease in common pochards from this HPAI H5N8 virus in 2014 is in contrast to previous findings based on experimental inoculation of common pochards with HPAI H5N1 GsGd virus (clade 2.2.1)15, indicating both virus circulation in the population and the ability to survive infection. In addition, HPAI H5N8 GsGd viruses  were isolated from apparently healthy free-living Eurasian wigeons sampled in Russia in 201418, and in the Netherlands in 2014 and in 201519. Eurasian wigeons are strongly migratory, and make use of multiple major migratory flyways20. There are at least three wintering populations of Eurasian wigeons: Western Europe/Mediterranean/Africa, Northern India, and South-East Asia. The majority of these populations breed in northern Russia, and it is not clear to which degree their breeding areas overlap22.Of the four duck species investigated here, the Eurasian wigeon is the most likely candidate as long-distance vector of HPAI H5N8 GsGd virus as infection was not associated with disease and virus excretion was most abundant in this species. Further support for Eurasian wigeons as a long-distance vector of HPAI H5N8 virus comes from the detection of H5N8-specific antibodies in blood samples collected in Korea and the Netherlands24. Serological findings in free-living common teals sampled in Korea in 2014 provided evidence for survival of the infection with HPAI H5N8 virus , ancestral to HPAI H5N8 virus as detected in Europe in 20148. Common teals are strongly migratory and their wintering populations cover a large range: southern Europe, southern Asia, and Africa25. Despite lower virus excretion, this species cannot be excluded as a long-distance vector for HPAI H5N8 virus.In common teals, HPAI H5N8 virus excretion was lower than that in Eurasian wigeons. The virus replication, as found here in common teals, is in line with the findings in live birds sampled in Europe and Korea in 2014. HPAI H5N8 virus was detected in a swab from an apparently healthy common teal sampled in Germany in 2014 (sample type not specified)13. Nevertheless, HPAI H5N8 GsGd viruses had been detected in hunter-harvested apparently healthy mallards sampled in Germany in December 2014 and January 201526. Ancestral to HPAI H5N8 virus as detected in Europe in 20148. Mallard wintering populations range from Europe to Southeast Asia25. Hence, mallard as a species may act as long-distance vector—either directly, or indirectly via linked shorter-distance migratory populations27—for HPAI H5N8 viruses.HPAI H5N8 virus excretion was relatively low in mallards, which was suggested as a prime candidate for long-distance vector of HPAI H5N1 virus15 and could therefore not be compared with our findings. Common pochards are migratory and winter in southern Europe, Africa, and southern Asia25. The absence of disease from this HPAI H5N8 virus in 2014 is in contrast with the previous findings from HPAI H5N1 virus, based on experimental inoculation of common pochards with HPAI H5N1 GsGd virus (clade 2.2.1), which resulted in mild to severe neurological disease13, and the detection of HPAI H5N1 GsGd virus in free-living common pochards found dead in Europe  in 2006 (clade 2.2/2.2.1)). At that time, the virus also was detected in one apparently healthy common pochard28.In addition to the dabbling ducks, the diving duck species investigated here—common pochard—became infected without evidence of the disease. In 2014/2015, HPAI H5N8 virus was not detected in common pochards sampled alive or found dead in Europe or Asia. However, in total, just one live bird and no dead birds were sampled in the field30. Based on a combination of RT-PCR, virus isolation, and IHC, the respiratory tract is the most likely source for the virus excreted through the pharynx. In chickens, a combination of RT-PCR, virus isolation, and IHC implied systemic virus replication (Table Anas strepera) (at 4 dpi13), mallard , and tufted duck (Aythya fuligula) (at 3 or 4 dpi16). In our study, the lack of evidence for HPAI virus replication in the intestinal epithelium at 4 dpi corresponds with the absence of infectious virus excretion from the cloaca at 4 dpi in 16 birds from which the tissues were taken. Most ducks excreted infectious HPAI H5 virus from the cloaca at 3 dpi ; therefore the time point for IHC at 4 dpi may have been too late to identify the location of virus replication. However, based on virus isolation from tissues and evidence of virus replication by IHC at 4 dpi, the kidney, pancreas, and/or liver are the most likely source for virus excreted through the cloaca. This is in contrast to most LPAI viruses, for which the epithelium lining of the intestinal tract is the main replication site33. Thus, also for this HPAI H5N8 virus, the main route of excretion in ducks seems to be from the pharynx.The pattern of HPAI H5N8 virus infection in the duck species investigated here was similar to that shown previously for HPAI H5 GsGd virus infection in dabbling ducks and diving ducks. Similar to earlier findings in dabbling and diving ducks, HPAI H5 virus excretion from the pharynx was higher than that from the cloacaon Table -3. Despi13, northern pintail (Anas acuta)12, and mallard29 (Table 13. This suggests that HPAI H5N8 GsGd virus (clade 2.3.4.4 group A) has a lower virulence for common pochards than HPAI H5N1 GsGd virus. Nevertheless, in 2016/2017 HPAI H5N8 GsGd clade 2.3.4.4 viruses of a different genetic group 8 emerged in wild birds and poultry in Europe associated with mass die-offs among wild birds (including Eurasian wigeons)34. Experimental infection of wild birds, according to a similar protocol as above, would allow evaluation of possible changes in the susceptibility to infection and virulence of HPAI H5 GsGd clade 2.3.4.4 group B compared to group A.Experimental infections with HPAI H5 GsGd viruses show differences in virulence within and between different duck species. In dabbling ducks, no evidence of disease was shown as a result of HPAI H5N8 GsGd virus (clade 2.3.4.4 group A) infection, as has been shown previously for HPAI H5N1 GsGd virus infections in subadult dabbling ducks of the species gadwall, Eurasian wigeon, common teal29 Table . In diviThe results of this study provide the strongest evidence for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus (clade 2.3.4.4 group A), although common teals, mallards, and common pochards cannot be excluded. This switch in level of virus excretion—highest in Eurasian wigeons for the 2014 virus, compared to highest for common pochards and mallards for the 2005 virus—and the decreased severity of clinical disease—no clinical disease in any of the birds infected with the 2014 virus, compared to over 50% clinical disease and pathological changes in common pochards infected with the 2005 virus—implies that the HPAI H5 GsGd viruses have adapted to wild waterbird populations over time. Whether this means that the observed changes allow more efficient long-distance virus transport in Eurasian wigeons is yet to be determined. Also, only a very limited number of waterbird species were examined in this study, and it cannot be excluded that other waterbird species are involved in HPAI H5 epidemiology. Further studies that combine laboratory evaluation of HPAI viral virulence and transmissibility in wild waterbirds and field studies on the spread of HPAI virus in wild waterbirds, as well as on the spatial and temporal connectivity between their migratory flyways, are essential to clarify the changing role of wild waterbirds in the epidemiology of HPAI H5 GsGd virus infections.7.3 median tissue culture infectious dose (TCID50)/ml and was diluted with phosphate-buffered saline (PBS) to 1 × 104 TCID50/3 ml. This virus was chosen due to its high pathogenicity in naturally infected chickens and for consistency to compare the findings with ferrets experimentally infected intranasally with the same H5N8 virus35. All experiments with HPAI H5N8 virus were performed under Animal Biosafety Level 3 conditions.The HPAI H5N8 virus of the clade 2.3.4.4 used in this study was isolated from the lung homogenate of a naturally infected chicken during an outbreak at a farm in Ter Aar, the Netherlands, in 2014 (A/chicken/Netherlands/emc-3/2014). Full-genome sequence of this virus is available in the GenBank under the following accession number: KR233687 to KR233694. The virus was propagated by two passages in Madin–Darby canine kidney (MDCK) cells. The harvested supernatant had a titer of 1 × 10Aythya), mallard, common teal, and Eurasian wigeon . For each species, males and females were equally represented. All ducks used for the infection experiment were captive-bred . Both chicken and ducks arrived in the lab 11 days prior to the day of inoculation, were in good condition, and looked apparently healthy. Birds were moved into isolators 3 days prior to the day of inoculation. Ducks were approximately 1 year of age at the time of inoculation. Blood samples (−3 days post inoculation [dpi]), cloacal swabs, and pharyngeal swabs (−3 dpi and 0 dpi) were collected from all ducks before inoculation. Serum was analyzed for the detection of antibodies specific to influenza A virus , using a commercial blocking enzyme-linked immunosorbent assay (bELISA)  and according to the manufacturer’s instructions. Swabs were analyzed for the detection of influenza A viruses by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the matrix gene. No NP-specific serum antibodies were detected. All ducks were tested negative for virus detection by RT-PCR for the matrix gene, except for three ducks . These birds tested weakly positive for virus by RT-PCR for the matrix gene  and one of the two common teals tested weakly positive for H5 virus detection by RT-PCR . No influenza A viruses were isolated from these samples upon inoculation into the 11-day-old embryonated chicken eggs, all three ducks subsequently became infected with HPAI H5N8 virus after inoculation, and two of these three ducks showed the highest virus excretion of their species. Birds had access to food and water ad libitum. Prior to the experiment, ducks were housed together in one animal room. A total of four specific-pathogen-free White Leghorn chickens of 14 months of age at the time of inoculation were used as positive controls for the pathogenicity of the virus stock. Chickens were obtained from the Animal Health Service, Deventer, the Netherlands. Neither NP-specific antibodies by bELISA, nor influenza A viruses by RT-PCR for the matrix gene were detected in chickens prior to the experiment at −3 and 0 dpi.Ducks of four different species were included in this study: common pochard  at 2–3 dpi—not related to the HPAI H5N8 virus infection, but caused by trauma—we decided to euthanize these two birds at 4 dpi instead of at 18 dpi, as initially determined by randomization. Therefore, the common teals were no longer randomly divided into two groups of birds. In addition, two birds—both supposed to be euthanized at 18 dpi—had to be removed from the experiment at 10 dpi due to lethargy (Eurasian wigeon no. 8) or at 11 dpi due to luxation of the left leg (common pochard no. 16). No viruses were isolated from the tissues and cloacal or pharyngeal swabs of these birds at the day of removal from the experiment.As expected, by 2 dpi 100% of the positive-control chickens were dead. In addition to comparing the findings of experimentally infected ducks with those of experimentally infected chickens, we also compared them with the findings of five H5N8-positive chickens found dead that were collected during the same outbreak as from which the virus strain A/chicken/Netherlands/emc-3/2014 (H5N8) was isolated. The study was approved by an independent animal experimentation ethical review committee, approved by the Dutch government (Stichting DEC consult) (permit number 122-15-01 and 122-15-02).37. Briefly, RNA from the tissue and swab suspensions was isolated using a MagNaPure LC System with the MagNaPure LC Total Nucleic Acid Isolation Kit . RT-PCR were performed on an ABI 7700 machine  using a TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). The oligonucleotides (5′-CTT-CTR-ACC-GAG-GTC-GAA-ACG-TA-3′) and (5′ -TCT-TGT-CTT-TAG-CCA-YTC-CAT-GAG-3′) and the labeled probes (5′ 6-FAM-TCA-GGC-CCC-CTC-AAA-GCC-GAG-A-BHQ-3′) and (5′ 6-FAM-TCA-GGC-CCC-CTC-AAA-GCC-GAA-A-BHQ-3′) were used for detection of the matrix segment of influenza A viruses. Samples were considered virus positive if the cycle threshold (Ct) value was <40.Tissues collected for virus detection and titration were the brain, trachea, lung, air sac, pancreas, liver, jejunum, colon, heart, spleen, and kidney. Tissues were weighed and homogenized in 1 ml virus transport medium using a homogenizer . RNA isolation and RT-PCR of the tissue samples and cloacal and pharyngeal swabs were performed as described previously38. To compare the virus culture in MDCK cells and in embryonated chicken eggs, aliquots of a cloacal swab and a pharyngeal swab collected from each of the eight Eurasian wigeons at 3 dpi were inoculated in parallel into the MDCK cells and into the 11-day-old embryonated chicken eggs. All 16 samples were tested positive in the matrix-RT-PCR . From three of the eight Eurasian wigeons, no virus was cultured using either method; from the remaining five Eurasian wigeons, virus was cultured from six of the ten samples using both methods, and the virus was cultured from another two samples exclusively using embryonated chicken eggs.Virus titers were shown as per gram tissue and determined in triplicate based on 10-fold serial dilutions of the homogenized tissue samples and swabs on MDCK cells, as described39.At 4 dpi, the samples of the following tissues were collected from ducks for histopathology and IHC: nasal turbinates, brain, trachea, lung, air sac, pancreas, liver, esophagus, proventriculus, duodenum, jejunum, ileum, colon, cecum, heart, spleen, kidney, adrenal glands, gonads, bursa, and pectoral muscle. At 1 or 2 dpi, the same tissues plus oviduct and wattle were collected from chickens. For the naturally infected chickens, the same tissues were collected, except for the pectoral muscle, gonads, adrenal, duodenum, and ileum. The tissue samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4-μm-thick sections. For histopathologic examination, the tissue sections were stained with hematoxylin and eosin (HE). For detection of cells expressing influenza A virus antigen, the tissue sections were subjected to IHC analysis with a monoclonal antibody against the nucleoprotein of influenza A virus as primary antibodyBody mass was monitored daily from 0 to 10 dpi, and on 12, 14, 16, and 18 dpi. Each morning before handling the birds, the individual birds in each group were scored quantitatively for nervous and other clinical signs. Nervous signs included walking toward a person, unsure tread, circling, falling over, tremors, and torticollis. Other clinical signs included ruffled feathers, decreased movement or feeding activity, exudate from eyes or nose, labored breathing, and lethargy.t-test or Wilcoxon test was performed. To investigate whether the differences in virus excretion or duration were statistically significant among species, a one-way ANOVA or Kruskal–Wallis test was performed.To compare virus excretion among and within groups, the AUC of infectious virus (based on virus isolation) or viral RNA (based on RT-PCR) from 0 to 4 dpi was calculated. The mean quantity of virus excreted per group from the cloaca or pharynx  was based on the AUC of all birds in the group. To compare the duration of virus excretion among and within groups, the median of maximum day of infectious virus presence  was used. The median duration of virus excretion per group was based on the values of all birds in the group. To investigate whether the differences in virus excretion or duration were statistically significant between routes of virus excretion , a paired Figure S1 Body mass of individual ducks in time . Eurasian wigeon (A); common pochard (B); mallard (C); common teal (D)50/mlFigure S2 Virus excretion of individual birds based on virus isolation of highly pathogenic avian influenza virus A/chicken/Netherlands/emc-3/2014 (H5N8) GsGd clade 2.3.4.4  via the pharynx  and via cloaca , with minimal detection limit of 0.5 TCIDFigure S3 Virus excretion of individual birds based on RT-PCR detection of highly pathogenic avian influenza virus A/chicken/Netherlands/emc-3/2014 (H5N8) GsGd clade 2.3.4.4  via the pharynx  and via cloaca Table S1Table S2Table S3Table S4


To investigate the costs of treatment with basal insulin , and premixed insulin (PM) in routine clinical care.Cohort study based on data from the Swedish National Diabetes Register, including 5077 insulin-naïve men and women with type 2 diabetes, resident in a distinct geographical region of Sweden. Patients were included between 1 July 2006 and 31 December 2009 and followed for 12 months. All drug- and healthcare-related costs, stratified by diabetes-related or non-diabetes care contacts, were quantified and compared to baseline.Initiation of insulin treatment generally entails increased diabetes-related health care contacts and treatment costs, and decrease in health care costs. The median changes in costs were generally smaller than the mean changes, reflecting great variations between patients. The treatment costs were higher for IG, ID and PM compared with NPH, although higher age, history cardiovascular disease and diabetes complications as well as higher diabetes-related and other treatment costs were independent predictors. Overall, only PM (but not IG or ID) were associated with higher diabetes-related health care costs, although these were also independently predicted by cardiovascular morbidity and markers of complicated diabetes.This study demonstrates that the initiation of insulin in patients with type 2 diabetes in clinical practice leads to increased health care contacts, overall and treatment costs, but also generally results in a decrease in health care costs. The diabetes-related treatment cost was lowest using NPH insulin but only premixed insulin was associated with higher diabetes-related health care costs than NPH.The online version of this article (doi:10.1186/s40842-015-0017-1) contains supplementary material, which is available to authorized users. Insulin treatment is commonly used in patients with type 2 diabetes when lifestyle changes and oral hypoglycemic agents (OHA) fail to achieve adequate glycemic control , 2. The The clinical effects of these treatment alternatives have been evaluated in randomized clinical trials (RCT) and meta-analyses, and recently also in clinical practice , 5. OverThe high costs associated with diabetes care have long been recognized, but the number of published studies in this area is low. A recent report based on managed care administrative data in the U.S.A. described in detail the total health care costs for patients with type 2 diabetes . The strThere are no studies available comparing costs of different insulin regimens when added to OHA in unselected cohorts in clinical practice. The aim of this study was therefore to examine health care utilization and costs in our recent study with 5,077 insulin-naïve type 2 diabetes patients, after starting treatment with NPH, IG, ID or PM .The overall design of this study has recently been described in detail . To summAll included patients have agreed by informed consent to be registered before inclusion. The Ethics Review Board at the University of Gothenburg approved the study.We included insulin-naïve patients with type 2 diabetes, at least 18 years of age. The study period was between 1 July 2005 and 31 December 2010. Patients were not allowed to fill a prescription of insulin from 1 July 2005 to 30 June 2006 to ensure they were previously untreated (data from the Prescribed Drug Register). Patients were required to have their first prescription of insulin filled (index date) between 1 July 2006 and 31 December 2009 to allow for 1 year of follow-up. Patients were thus followed for 12 months or until the occurrence of a censoring event. The mean number days of follow-up were similar in the four treatment groups  [Treatment costs (drug-related costs) were retrieved from the Prescribed Drug Register, which has full, nation-wide coverage of all transactions that are made at pharmacies in Sweden, including all drugs (ATC codes A-V) and technical aids (ATC codes W-Y). We used data between 2005-07-01 and 2010-12-31.Healthcare costs were estimated by using the associated DRG-codes (data from VEGA). To make data comparable, Swedish national DRG-weight lists and costs for 2010 were used . VEGA also provided data on outlier costs, which were considered when estimating the final cost per care contact. For outdated DRG-codes, old DRG-weight lists provided by the Swedish National Board of Health and Welfare were used to extract weights, which were used in combination with the 2010 DRG cost. Further, psychiatric DRG-codes were flagged, and the clinic setting where the care contacts occurred, were used to determine whether DRG-weights from the inpatient DRG-list or the psychiatric DRG-list should be applied. For primary care, costs were determined based on the caregiver, where physicians were assumed to have a three times higher rate than other caregivers . This corresponded to a 1840 SEK cost for a physicians visit, and a 615 SEK cost for all other primary care visits. The approximate value of 100 SEK is currently 12 US dollars (August 2015).www.scb.se/en).We studied all drug- and health care-related costs, stratified by diabetes-related or non-diabetes care contacts. All costs 12 months before initiation and during follow-up of insulin treatment were recorded. A correlation analysis with costs before and after insulin treatment initiation was performed, to determine whether incremental analysis or pre-index cost adjustment should be undertaken. If the correlation exceeded 0.5, an incremental analysis whereby the prior (baseline) costs are subtracted from the costs observed during follow-up, was recommended. Otherwise, the pre-index costs were included as a covariate in the regression model. Costs are presented in 2010 SEK value after adjusting for consumer price indices provided by Statistics Sweden . We also identified all diabetes-related care contacts and their associated costs through the ICD-10 codes E11*, E13*, and E14* (regardless of diagnosis position), as well as cardiovascular disease costs, such as myocardial infarction/ischaemic heart disease (I20-I25), atrial fibrillation (I48), congestive heart failure (I50*), and stroke .χ2 test. For continuous variables with non-normal distribution a Kruskal-Wallis test was performed. All continuous outcome variables were explored for their distribution.Baseline characteristics are presented as means ± 1 standard deviation (SD) or medians for continuous variables and frequencies for categorical variables with crude significance levels for differences between the groups, when analysed using ANOVA or We used generalized linear modeling (GLM) after log transformation to assess potential predictors of diabetes-related health care contacts, health care costs and treatment costs. For each outcome, three models were explored and presented; firstly, unadjusted where outcome was as a function of insulin groups, secondly, including covariates with few missing values , and finally a fully adjusted model including age, gender, level of income, diabetes duration, history of CVD, history of diabetes complications, previous OHA use, follow-up time, pre-index HbA1c, pre-index BMI and weight. Pre-index diabetes-related costs were included as a covariate to account for the patient’s type 2 diabetes history and severity. Further, post-index other costs (including cardiovascular costs) were included in the model as a covariate to account for the overall disease burden (comorbidities) of the population, which in turn might affect post-index diabetes-related costs.p value <0.05 was considered statistically significant.Statistical analyses were performed in R  or SAS V.9.3 . A two-sided As previously reported, 5077 insulin-naïve patients with type 2 diabetes were included in the study . The patThe numbers of visits in health care before and after starting the insulin treatment are given in Table The costs of health care and treatments before and after starting the insulin treatment are given in Table For diabetes-related costs where also cardiovascular costs were included Table , similarThe median costs for both health care and treatment in both Tables p < 0.01). Overall, the differences in incremental other costs between insulin groups were non-significant, while the patterns indicated a moderate increase for treatment costs for all groups.For other pre-index health care costs Table , the PM For patients who did have a change in diabetes-related health care costs (N = 4232), IG and PM increased costs compared with NPH, whereas ID lowered costs in an unadjusted model  were associated with higher costs, although these were also independently predicted by cardiovascular morbidity and markers of complicated diabetes.There are generally differences in clinical characteristics between the patients offered the various treatment options when additional treatment is required on top of OHA in clinical practice. Still, this project including previously presented clinical results , provideOther studies of the costs of initiating basal insulin have reached similar conclusions. A retrospective study on a U.S. population between 2001 and 2006 found that IG compared with NPH had higher drug-related costs, but no significant difference in health-care costs, although both were associated with major cost reductions . SimilarThe present study has several strengths. The observational design allows for comparisons of the effectiveness and costs of different types of insulin, and the results are likely to be representative of clinical practice in countries with similar populations, following similar treatment guidelines . All patThis study demonstrates that the initiation of insulin in patients with type 2 diabetes in clinical practice leads to increased health care contacts, overall and treatment costs, but also generally results in a decrease in health care costs. The diabetes-related treatment cost was lowest using NPH insulin but only premixed insulin was associated with higher diabetes-related health care costs than NPH.


Phlebovirus and Tenuivirus; however, the predicted proteins encoded by RNA2 are related to the movement protein (MP) in the genus Ophiovirus and Emaravirus. Furthermore, these two viruses define a novel clade in the family Phenuiviridae, order Bunyavirales, which is phylogenetically related to the viruses in the above four genera. Moreover, after mechanical inoculation with WCLaV-1 seedlings of the natural host watermelon plants develop crinkling similar to those observed in the field. These findings enhance our understanding of the evolution and the classification of ssRNA viruses.Two novel negative-sense, single-stranded (ss) RNA viruses were identified in watermelon plants and named watermelon crinkle leaf-associated virus 1 and 2 (WCLaV-1 and -2), respectively. The multipartite genomes consist of three RNA molecules of ~6.8, 1.4, and 1.3 kb. The genomes and the deduced proteins of RNA1 and RNA3 show features resembling those of members in the genus  Citrullus lanatus) is an economically important fruit crop in many countries and is nutritionally beneficial to humans , a putative member of the genus Deltapartitivirus in the family Partitiviridae and two orthotospoviruses, watermelon bud necrosis virus (WBNV), and melon severe mosaic virus (MeSMV) in the family Tospoviridae have been detected in watermelon plants  established the new order Bunyavirales to accommodate nine families that comprise 13 genera . These viruses are a unique group that infect a wide range of hosts, including vertebrates, invertebrates, and plants , Phenuiviridae (Tenuivirus), and Tospoviridae (Orthotospovirus). Members of the order Bunyavirales have similarities in genome organization and particle morphology, e.g., spherical in emaraviruses and orthotospoviruses and filamentous in tenuiviruses , the Phenuiviridae, order Bunyavirales.In 2015 and 2016, during a field survey in Kāifēng, Hénán Province, China, virus-like symptoms on leaves of watermelon were observed. To identify potential causal agents, RNA sequencing (RNA-seq) and small RNA sequencing (sRNA-seq) were carried out. Here, we describe and discuss the molecular and biological properties of two new (-ss) RNA viruses that were discovered from the infected leaves, named watermelon crinkle leaf-associated viruses 1 and 2 (WCLaV-1 and -2), as possible representatives of a novel taxon in the family In July 2015, leaf samples with mosaic and curling symptoms were collected from watermelon plants of cv. Hēixiùlóngjǔanfēng grown in the field in Hénán Province, China. Subsequently, in June 2016, watermelon samples with virus-like symptoms were randomly collected from Kāifēng County, Hénán Province. Samples were stored at −70°C.de novo assembly with a k-mer of 17 for small RNA data  and healthy leaves (sample KF-0) were selected for small RNA-sequencing (sRNA-seq) in 2015; leaves coded KF-15 were used for RNA-sequencing (RNA-seq) in 2016. Total RNAs were extracted using TRIzol reagent  following the manufacturer's protocol but modified to repeat the chloroform extraction and subsequent centrifugation step ; (II) identification of conserved and functional domains of the predicted proteins in WCLaV-1 and WCLaV-2 using the Conserved Domain Database (CDD) in NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)  ; (V) multiple sequence alignments first using the Clustal W method and then MEGA 6.0  prediction of ORFs using the ORF Finder program in NCBI  infected by WCLaV-1 and homogenized in 10 mL 0.01 M phosphate buffer (PB) (pH = 7.2), was used for mechanical transmission tests on four-leaf-stage seedlings of watermelon cv. Hēixiùlóngjǔanfēng. Plants non-inoculated and inoculated only by PB were used as negative controls. After inoculation, the seedlings were maintained in a clean growth chamber at 25°C and 80% relative humidity with 16-h light/8-h dark. The mechanically inoculated plants were carefully observed for symptoms and subsequently tested for virus infection by RT-PCR assays with the virus-specific primer pairs listed in Table de novo assembly and BlastX and BlastN searches against nr and nt databases, ZYMV (genus Potyvirus), WMV (genus Potyvirus), melon aphid-borne yellows virus , CiLCV (genus Deltapartitivirus), and four contigs homologous to viruses in the genera Phlebovirus and Ophiovirus were identified in sample KF-1 (Table Tobamovirus), watermelon virus A , and three contigs homologous (high similarity with that identified in KF-1) to phleboviruses and ophioviruses in the sample KF-15 collected in 2016 .The genomes were obtained by RT-PCR and RACE-PCR Figure  using viPhlebovirus , but was not found in WCLaV-1  and WCLaV-2 shares 27.2% aa sequence identity with rice stripe virus , both remarkably higher than with viruses in other genera, but far lower than the 75% identity between these two viruses.RNA1 for both viruses is the largest segment and has only one ORF (ORF1): 6,636 nt and 6,680 nt for WCLaV-1 and -2, which start, respectively, at position 6,584–6,586 and 6,634–6,636 and end at position 38–40 and 40–42. ORF1 encodes a putative RNA-dependent RNA polymerase  of 2,182 amino acids (aa) and 2,198 aa with a predicted molecular weight of 252.76 and 253.63 kDa, respectively, for WCLaV-1 and -2 Figures . A Blastus Table . Additious Table . Interess Figure . Both WCs Figure , was alss Figure . In addis Figure , which as Figure . MoreoveE = 1.4) with the 54 kDa protein of citrus psorosis virus , demonstrating a putative movement protein (MP) function, for WCLaV-1 and 19% identity  to the 58 kDa MP of blueberry mosaic associated virus (BIMaV) in the genus Ophiovirus   and WCLaV-2  may, respectively, encode a protein (p2) of 44.29 and 46.11 kDa, spanning position 1,356–1,358 and 1,366–1,368 (AUG start codon) to position 196–198 and 155–157 (UGA stop codon) of vcRNA Figures . Interescl05345), the typical domains in nucleocapsid proteins of viruses in the genera Tenuivirus and Phlebovirus, with a Pfame-value of 5.4D-16, 3.2e-17, respectively, distinct from those in other genera. Moreover, the highest amino acid identities for the nucleocapsid protein, 17.2 and 17.4%, respectively, were found with SALV and sandfly fever Naples virus  for WCLaV-1 and WCLaV-2, far lower than the 47.9% aa identity between these two viruses for nucleocapsid protein (Table Tenuivirus) for WCLaV-1 and Punta Toro virus  for WCLaV-2  of 347 a and 346 aa with a molecular mass of 38.75 and 38.68 kDa, respectively Figures . It is wIn addition, the highly conserved nucleotides 5′-ACACAAAG-3′ at the 5′-terminus were found in each RNA1 segment, and the conserved nucleotides 3′-UGUGUUUC-5′ at the 3′-terminus were detected in each RNA2 and RNA3 segment Figures . The 5′-Tenuivirus, Phlebovirus, Goukovirus, Phasivirus, the family Phenuiviridae  RNA viruses, we constructed phylogenetic trees based on the amino acid sequences of the putative RdRp Figure , MP Fig and NP . Accordie Figure . Howevere Figure . WCLaV-1The mechanical transmission experiment with WCLaV-1 was done three times with 20, 23, and 46 healthy watermelon plants inoculated, respectively. Each time, only one plant, respectively, of the inoculated samples was positive for WCLaV-1 infection by RT-PCR, thus conclusively showing infection rates of 5.0, 4.3, and 2.2%, respectively. For example, at 17 dpi, WCLaV-1 was detected only in watermelon plant No. 4 using RT-PCR with primer pair NP-1F/R, and the positive band was further identified by sequencing Figure . MoreoveThe development of NGS and bioinformatics has accelerated the discovery of novel viruses , as opposed to the GDNQ characteristic of non-segmented negative-stranded RNA viruses, are found in the putative RdRp of these two viruses  RNA viruses (except ophioviruses) generally contain a glycoprotein (GP) precursor, which has not been detected in WCLaV-1 and WCLaV-2 so far. Second, no conserved domains were found in the proteins encoded by RNA2, predicted as the MPs of WCLaV-1 and WCLaV-2, which are widely found in plant-infecting (-ss) RNA viruses  by RT-PCR and deep analyses of virus-derived sRNAs and contigs failed, we cannot exclude the potential existence of one or more additional genomic RNAs. Additionally, the sequences may include stretches due to cap snatching because the RACE was performed on total RNA.Orthotospovirus, family Tospoviridae) can be transmitted to healthy onions by mechanical inoculation in a controlled glasshouse at a relatively low transmission rate  RNA viruses, which are confirmed as members belonging to a previously undescribed novel clade in the family XW: conceived and designed the experiments. MX, WL, YR, and MC: performed the experiments. MX, WL, XZ, and MC: analyzed the data. XW, MX, and MC: wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arenaviruses and Coronaviruses (CoV) are enveloped RNA viruses. The nucleoproteins (N) of these viruses (termed nucleocapsid in CoV) play a key role in the formation of the viral ribonucleoprotein (RNP) complexes and in causing type I interferon (IFN) suppression. The primary function of the N proteins of CoV, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), and of arenaviruses, such as Lassa virus that can cause severe and lethal hemorrhagic fever infections in humans, is to package the viral single stranded genomic RNA(s) into RNP complexes , 2. AnotThe host innate immune system presents a significant barrier and defense against viruses. The host pattern recognition receptors (PRRs), including retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 protein (LGP2), can specifically recognize virus-specific components, such as RNA, DNA or glycoproteins. Following virus recognition by the PRRs, important cellular signaling pathways are activated to lead to production of cytokines, chemokines and type I IFNs, which play a critical role in the eradication of the virus. Arenaviruses and CoV both have evolved unique mechanisms to evade recognition by PRRs, which lead to type 1 IFN inhibition.in vivo through the activation of the RIG-I pathway [Immune stimulatory dsRNA generated during virus replication is important for RIG-I recognition and subsequently mediating type-I IFN production. We have previously discovered that arenaviruses encode a 3'-5'exoribonuclease function in the C-terminal domain of the N protein (NP) that acts as a type I IFN-antagonist [Coronaviruses are the only other family of viruses known to encode the DEDDh exoribonuclease in the N-terminal domain of their non-structural protein 14 (nsp14), which has been attributed to its important proofreading role during viral RNA genome replication . RecombiThe N protein of CoV has recently been found to employ a different way to mediate suppression of type 1 IFN production. Hu et al.  showed t


Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3’ poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3’ proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659.A new species of the family  Medicago sativa) is the most extensively cultivated forage legume in the world and the fourth most widely grown crop in the US, planted on more than 23 million acres in all 50 states. The estimated worth of alfalfa hay is over $8 billion annually. In addition to its value as livestock forage, alfalfa also has potential to make important contributions to bioenergy production. Alfalfa productivity is often limited by different diseases, insect pests and abiotic stress factors [Alfalfa , was not previously described in alfalfa or any other plant species.Alfalfa samples from four commercial pivot irrigated fields, 60 ha each, in Sudan were submitted for disease analysis. In total, nine different alfalfa samples designated 98.3A, 89.2AS, 89.3H, 89.1H, 89.1AS, 98.1A, 98.3A(0), 98.2RR and 97.2A were used in RT-PCR, dot blot hybridization and Western blot (WB) assays.For dot blot hybridization, RT-PCR assays and for Illumina RNA sequencing, total RNA was extracted from alfalfa samples using TRIzol RNA isolation reagent as described by the manufacturer (ThermoFisher Scientific). For dot blot hybridization, one μl of total RNA ~ 100–300 ng) was dotted directly onto a nylon membrane and cross-linked by ultraviolet light using a UVP HL-2000 HybriLinker  for three minutes. Hybridization (performed in UVP HL-2000 HybriLinker hybridization oven), washing and detection conditions were done exactly as described in the Boehringer Mannheim guide for filter hybridization [0–300 ng RT-PCR was performed with total RNA employing SuperScript RT-PCR system according to the manufacturer’s directions (ThermoFisher Scientific). Primers for RT-PCR are shown in RT-PCR products were either sequenced directly or cloned into pCRII-TOPO vector with dual promoter (ThermoFisher Scientific) for sequencing and synthesis of DIG-labeled RNA probes. DIG-labeled probes were synthesized using DIG RNA labeling mixture and T7 RNA polymerase (ThermoFisher Scientific) essentially as described .Library construction and high-throughput sequencing (HTS) on an Illumina Platform were outsourced and performed by SeqMatic Inc. . Contigs were assembled from the read data using CLC Genomics Workbench software version 9.5.2.The 5’end of the virus was determined using the 5-RACE system (ThermoFisher Scientific), PCR with several genome-specific primers , leaves were homogenized in sterile water followed by centrifugation of the extracts in a bench-top Eppendorf centrifuge for five minutes at 16.1 k rcf (13.2 k rpm). Virus captured on the TEM grids was stained with 1% phosphotungstate (PTA) solution. The grids were examined in a Hitachi H-7700 Electron Microscope at the Electron and Confocal Microscope Unit, Beltsville Agricultural Research Center.Alfalfa plants collected in Sudan and used in this work exhibited chlorosis and stunting. Upon arrival, the samples were partially deteriorated; visual signs and symptoms for identification of any possible diseases were not clear enough to be recognized. Preliminary rounds of RT-PCR (not shown) with total RNAs extracted from the tissues suggested that plant sample designated 98.3A might be infected with several viral diseases that commonly occur in alfalfa. Total RNA from this sample was subjected to HTS to detect all potentially present viruses in an unbiased manner.Cucumovirus, family Bromoviridae), and a virus with 90–97% identity to alfalfa enamovirus-1 , [Medicago sativa species, all host transcripts were assembled into a separate dataset and mapped onto the reference genome of cultivated alfalfa . The high percent identity (93–100%) confirmed the sample as alfalfa.Raw data reads were assembled into contigs and searched against a custom virus database by the translated BLAST tool (BLASTX) . Severalde-novo assembly of HTS-generated reads, 5’RACE and sequencing of the RT-PCR-amplified 3’terminus, consisted of 8,349 nucleotides excluding the 3’ poly(A) tail. NCBI BLAST searches revealed that the virus has its greatest sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. On the nucleotide level, the species most similar to the AVS were isolates A and B  of unclassified arachis pintoi virus [Alphaflexiviridae, blackberry virus E (BVE) [Allexivirus, ShVX [While PSV and AEV-1 have been reported in alfalfa , 13, AVSoi virus ; an unas E (BVE) , (50.6%)us, ShVX , (49.7%)us, ShVX .The complete AVS genome (GenBank accession № KY696659) contains six computationally predicted open reading frames (ORF), encoding the following putative proteins : ORF1, vpfam01660, nt 41–333, E-value 6.24e-72), 2OG-Fe(II) oxygenase , viral helicase 1,  and RNA dependent RNA polymerase . ORF1 also has domain hits with alkylated DNA repair dioxygenase AlkB , prolyl 4-hydroxylase alpha subunit homologues , and ATP-dependent exoDNAse .ORF1 of AVS (RdRp) has four conserved domains: viral methyltransferase, , with the TGB3 of the Escobaria virus, an unclassified member of the family Alphaflexiviridae (GenBank: AHB87054.1) and with the 8kDa protein of unclassified white ash mosaic virus (GenBank: ADI70511.1) [ORF4 is predicted to encode a TGB3-on codon ,20. The 70511.1) .Alphaflexiviridae (accession YP_009328895.1), 36% identity  with Garlic virus A, member of the genus Allexivirus (accession AGC09138.1) and 35% identity  with p42 of ShVX, a type member of the genus Allexivirus (NP_620651.1). This polypeptide is a distinctive feature of all known allexiviruses [ORF 5 (encoding unknown 38.4kDA protein) has 36% identity scores  with the 40kDa protein of arachis pintoi virus, unclassified member of the family iviruses ,20 and iiviruses .ACF37244.1), 49% identity  with CP of arachis pintoi virus A  with shallot mite-borne latent virus, unclassified allexivirus possibly synonymous with ShVX [ORF 6 (coat protein), has 46% identity  with the CP of ShVX , which are different alfalfa plants belonging to the same cultivar .Molecular data were also supported by immunological detection of the virus: polyclonal antibodies, produced against the peptide CP1 within the AVS CP specifically reacted with alfalfa samples 98.3A, 98.1A and less obviously with the sample 98.3A(0) in WB assay . The CP The original sequenced sample 98.3A used for HTS analysis and also two more samples, 98. 1A and 98.3A (0), that were later confirmed to be positive for AVS by dot blot hybridization and WB, were examined in a Hitachi H-7700 Electron Microscope. Filamentous virus particles about 600–800 nm in length and 13–16 nm in diameter were observed in the extracts from all tested samples . ShorterAllium spp. of monocotyledonous flowering plants, although some allexiviruses will induce local lesion on Chenopoidium species [Medicago sativa, legume family Fabaceae. The virus, tentatively named “alfalfa virus S” (AVS), is an unusual pathogen most closely related to arachis pintoi virus and BVE, which are putative and unclassified members of the family Alphaflexiviridae, respectively, and to several members of the genus Allexivirus. Even though the identity level between AVS and accepted allexiviruses is rather low, it has some of the characteristic features only found in allexiviruses, such as a serine-rich p38.4 protein of unknown function, as well as its general genome organization. Phylogenetic analyses clearly placed AVS together with arachis pintoi virus and BVE in a distinct cluster associated with the identified allexiviruses  indicates that AVS is a member of a novel, previously undescribed genus.We conclude that AVS represents a unique virus species with a general resemblance to the members of the genus


In humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs.Forty Nellore bulls were divided into three age groups: 1.8 to 2 years – young bulls; 3.5 to 7 years – adult bulls; and 8 to 14.3 years – aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA).p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05).We report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (These results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity. DNA damage in human spermatozoa has been associated with a variety of important outcomes including infertility , an incrSperm nuclear organization is very peculiar compared to somatic cells. It results from a long and complex process initiated during the late phases of spermatogenesis, essentially through spermiogenesis and completed when sperm cells go through the epididymis. In humans, it was reported that aging affect these processes resulting in a reduction in sperm chromatin organization which are associated with increased sperm morphological abnormalities and reduced motility. If the decrease in sperm quality associated with age is well documented it is not yet clear whether a certain immaturity of sperm structures and functions is suspected in young mammals. In young rats, Zubkova et al. (2005) reported a defect in protamination and a reduced level of disulphide-bridging on the cysteine-containing protamines when compared to adult animals . AlthougFor large domestic animals, with the help of genomic selection programs, there is a tendency to put into reproduction very young animals. Therefore, it is important to know whether young sexually mature animals present sperm characteristics that are at they optimum in terms of fertilizing capacity. Studies evaluating how aging may affect sperm chromatin integrity in bovine species and the source of the damage are scarce. One study in which semen quality in young bulls and again later in the same mature bulls were evaluated found consistent improvement in semen parameters including sperm DNA integrity, morphology and percentage of intact acrosomes upon aging . In a moBos indicus bulls (Nellore) were selected from an artificial insemination center located in the southeast of Brazil . The bulls were kept on their native pasture (Cynodon plectostachyus) and were given dietary supplementation to optimize their energy balance. Three ejaculates were collected from each bull using an artificial vagina according to a regular twice-a-week collection schedule between September and February of 2010. The samples were cryopreserved  in liquid nitrogen until the evaluation. The 40 selected animals were divided into three groups according to their ages at collection time: 1.8 to 2 years , 3.5 to 7 years , and 8 to 14.3 years .A total of 40 Flow cytometry analysis was performed on an Attune® apparatus  equipped with 488 nm and 405 nm laser beams and the following emission filters: BL1 530/30 nm “band pass” (BP), BL2575/24 nm BP, BL3 640 nm “long pass” (LP), and VL1 450/40 nm BP, VL2522/30 nm BP, and VL3 603/48 nm BP.Frozen semen samples (250 μl straws) were thawed at 35 °C for 20 s. Microscopic evaluations were performed using an Olympus BX61 microscope  equipped with phase contrast, differential interference contrast (DIC), and epifluorescence. The Computer Assisted Semen Analysis of sperm movement  used the IVOS “hardware” . The following kinetic spermatic parameters were evaluated in 2 μl sample aliquots on a slide with four chambers : MOT , MP (progressive motility), VAP (average path velocity), VSL (straight-line velocity), and VCL (curvilinear velocity). The fields were randomly selected for each analysis based on the absence of artifacts such as bubbles, particles, or other elements that could adversely affect the evaluation.Sperm alterations were classified according to Blom (1973)  and Bartper batch was thawed (35 °C/20 s). The semen sample was then diluted to 2.106 sperm in 200 μl of PBS (phosphate buffer solution) and stained with 3 μL (14 μM) of propidium iodide (PI) and 50 μL (12.5 μM) of Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (FITC-PSA). Samples were incubated at 37 °C for 8 min in the dark and analyzed in an Attune® flow cytometer with a 488 nm argon ion laser excitation beam and simultaneous BL1 (530/30) and BL3 (640LP) readings. The positive control semen samples were subjected to three cycles of “Flash Frozen”  staining) and acrosome integrity (via PSA-FITC staining) were considered together, the aged group presented the lowest percentage of undamaged cells, while the young animals had the highest percentage. The adult group presented intermediate values (Table p < 0.05). As shown in Table Table es Table . Finallyp < 0.05). Since CMA-3 staining is inversely correlated with the level of sperm nuclear condensation, this observation suggests that the sperm nucleus of young bulls is significantly less condensed than the sperm nucleus of adult and aged animals. When investigating DNA fragmentation indirectly using the acridine orange SCSA staining assay we found that both the young and the aged groups show the highest DNA fragmentation index (DFI) while the adult group was significantly lower (Table Table Bos Taurus bulls sperm motility decrease upon aging [Bos Indicus) [Our observations suggest that young bull sperm samples are characterized by their high motility and that upon aging motility parameters decrease. This is not new, since there are similar reports showing that on aging . It was on aging . Similaron aging , 23. It Indicus) –26. LookIndicus) , 28 whenOur combined evaluation of sperm plasma membrane/acrosomal membrane integrity showed a regular decrease upon aging. Since we dealt with cryopreserved samples this may be a reflection of their weaker ability to sustain the freezing and thawing protocol that are known to affect internal and external cell membranes essentially through the vicious circle of membranous lipoperoxidative processes . The ageTaken together, and, without surprise, progressive motility, functional mitochondria as well as plasma/acrosomal membrane integrity were found negatively correlated with age. Finally, when it came to sperm morphological alterations our study revealed that the highest percentages of structurally abnormal sperm were found for the young and the aged animal groups. This observation somewhat supports the idea that young bulls may be in a certain state of immaturity evidenced by this higher level of teratospermia.To analyze more deeply bull sperm characteristics in these three age groups we looked at the sperm nuclear compartment by evaluating sperm DNA compaction, sperm nuclear protamination and sperm DNA oxidation. The rationale behind these investigations was that if abnormal head morphology was a characteristic of young bull spermatozoa this might be due to nuclear alterations as it is supported by several studies –34. To oIn conclusion, our study brings forward that young bulls as well as aged bulls present spermatozoa that are not at their optimal condition in terms of nuclear integrity. In young bulls, deficient protamination and higher DNA fragmentation reveal a state of nuclear fragility or immaturity. In elderly bulls, higher susceptibility to oxidative alterations may explain their intermediate susceptibility to acidic treatment (DFI%) revealing also a certain state of fragility of the sperm nucleus. Although this study does not bring evidence as to the fertility potential of these 3 groups of bulls, which would be an interesting parameter to have, it has the advantage to show that both very young bulls sperm may not be at their optimal in term of nuclear integrity. This is particularly important to take into account because the paternal chromosomal lot is the essence of what must be conveyed by the spermatozoa in the oocyte. An intact paternal genome together with an intact maternal counterpart will drive the developmental program and will determine the quality of life of the offspring. In an industrial perspective, to know that young bulls, although sexually mature by some aspects, do show sperm cells that may be not totally mature is rather an important parameter. It may help to explain the lower reproductive efficiency of some bulls that are used as reproducers too early. It may also avoid misleading conclusions regarding the fertilizing ability of some bulls that may have been tested too early. In fine, based on our observations we propose that it might be of interest to introduce assays aimed at evaluating the integrity of the bull sperm nucleus together with the classical sperm evaluation check-list which includes sperm count, motility and morphological assessments. The results from this study suggest that the early introduction of young animals into artificial insemination centers should be cautiously evaluated because, despite providing satisfactory semen samples according to the traditional evaluation parameters, young animals may harbor a certain degree of nuclear/chromatin immaturity.


The complete genome sequences of 5 foot-and-mouth disease viruses of serotype A are reported here. These viruses originate from outbreaks in northern Nigeria in 2013 to 2015 and belong to the A/AFRICA/G-IV lineage. Aphthovirus (family Picornaviridae) that causes a highly contagious vesicular disease in cloven-hooved animals. The virus is classified into 7 immunologically distinct serotypes , each containing numerous genetically and geographically distinct evolutionary lineages or topotypes (2Foot-and-mouth disease virus (FMDV) is a positive-sense single-stranded RNA virus of the genus potypes 24. A recepotypes 2. In 2017potypes 2, 7 and Tpotypes 2. Here, wAll samples were collected from cattle herds located in northern Nigeria between 2013 and 2015. Total RNA was extracted from cell culture supernatant with a NucleoSpin RNA virus kit (Macherey-Nagel) and treated with Baseline-ZERO DNase (Epicentre) to remove host DNA. cDNA was synthesized according to the manufacturer’s instructions using SuperScript IV reverse transcriptase (Thermo Fisher Scientific), an anchored oligo(dT) primer, and an FMDV-specific internal primer. Sequencing libraries were prepared using a Nextera XT kit (Illumina) as described in the user’s manual. The fragment length distributions of the libraries were verified on a Bioanalyzer system (Agilent Technologies), and libraries were quantified using a library quantification kit (Kapa Biosystems). Sequencing was performed at the Biotechnology and Molecular Biology Platform  using a MiSeq reagent kit version 3 (Illumina) with 2 × 300-bp paired-end sequencing on a MiSeq system (Illumina).De novo assembly of the L fragment was performed using Iterative Virus Assembler (IVA) version 1.0.8  tract could not be determined. The genomes were predicted to contain a single open reading frame (ORF) of 6,990 to 6,996 nucleotides encoding a polyprotein of 2,329 to 2,331 amino acids.MG725872 to MG725876.The complete genome sequences for FMDV A/NIG/1/13, A/NIG/1/15, A/NIG/3/15, A/NIG/5/15, and A/NIG/6/15 have been deposited in GenBank under the accession numbers


Hyalomma spp. are considered the most competent virus vectors. CCHFV evades the tick immune response, and following its replication in the lining of the tick's midgut, it is disseminated by the hemolymph in the salivary glands and reproductive organs. The introduction of salivary gland secretions into the host cells is the major route via which CCHFV enters the host. Following an initial amplification at the site of inoculation, the virus is spread to the target organs. Apoptosis is induced via both intrinsic and extrinsic pathways. Genetic factors and immune status of the host may affect the release of cytokines which play a major role in disease progression and outcome. It is expected that the use of new technology of metabolomics, transcriptomics and proteomics will lead to improved understanding of CCHFV-host interactions and identify potential targets for blocking the CCHFV transmission.Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by bite of infected ticks or by direct contact with blood or tissues of viremic patients or animals. It causes to humans a severe disease with fatality up to 30%. The current knowledge about the vector-host-CCHFV interactions is very limited due to the high-level containment required for CCHFV studies. Among ticks,  Bunyaviridae) circulates in nature in an enzootic cycle between ticks and non-human vertebrates and poses a significant public health threat due to its high pathogenicity to humans. Humans are infected by bite of infected Ixodid ticks , or by contact with blood or tissues of viremic patients or animals. The disease (CCHF) is characterized by abrupt onset of fever, headache, fatigue, and myalgia, as well as gastrointestinal symptoms, such as nausea, vomiting, and diarrhea. Severe cases present hemorrhagic manifestations ranging from petechiae, epistaxis, ecchymosis, and gingival hemorrhage to severe hemorrhages from various systems. The fatality rate is up to 30%. Wild and domestic animals present a short viremia (2–15 days) and they do not develop clinical illness.Crimean-Congo hemorrhagic fever virus , medium (M), and large (L) segments which encode the nucleocapsid (N) protein, the glycoprotein precursor (which gives rise to the envelope glycoproteins Gn and Gc) and the L protein, respectively. The CCHFV genome is encapsidated by multiple copies of N protein to form a ribonucleocapsid complex which is critical for the virus replication cycle  which are the main competent vectors of the virus. The term “vector competence” is used to describe the ability of a vector to acquire, maintain and transmit a pathogen. H. marginatum is present in southern Europe and some parts of Asia and Africa. It is characterized by its aggressiveness in seeking human hosts. In CCHF endemic areas, where the climatic and environmental factors are suitable for H. marginatum ticks , their population is increased in spring and summer, accounting for >30% of tick species in the area. CCHFV has been detected or isolated from additional tick species, but studies are needed to show whether they are competent virus vectors, or merely coincidental unmaintained tick infection from recent feeding on an infected animal or co-feeding (feeding on an uninfected vertebrate host in close proximity with an infected tick) or the result of a recent blood meal on an infected animal. For an arthropod to be incriminated as an actual vector, several criteria must be met; such as vector competence in laboratory studies, and evidence that the arthropod species feeds in nature on a host that develops an appropriate viremia and that it is active at the time of the year that viral transmission is occurring  of the host. The co-evolution of the ticks, hosts and pathogens results in conflict or cooperation between them, benefiting ticks and pathogens and, to a lesser extent, hosts , and, less efficiently, by venereal transmission   antiviral response via the cellular adaptor MAVS (Spengler et al., Soon after the presentation of CCHFV antigen to host cells, innate and adaptive immune responses are activated Figure . DC-SIGNSeveral cytokines and chemokines are released during the course of CCHF, especially in severe cases (Ergonul et al., in vitro, offering an alternative approach to understand the way that tick cells respond to virus infection (Bell-Sakyi et al., in vivo transmission model for CCHFV in a BSL4 biocontainment was established recently using interferon knockout mice, which is an additional tool to study the transmission and interaction of CCHFV with its tick vector (Gargili et al., Tick cell lines are now available to enable the CCHFV studies in vitro models to increase our understanding of CCHFV pathogenesis for the good of Public Health.Over the last decades considerable progress has been made in the identification of the cellular components involved in tick-host-pathogen interactions. However, there is limited knowledge so far in the case of CCHFV due to the high-level containment required for studies with the virus. The identification of the molecular drivers that promote CCHFV survival in the tick, persistence and pathogen transmission provides the opportunity to disrupt these processes and lead to a reduction in tick burden and prevalence of tick-borne diseases (De la Fuente et al., AP wrote the first draft of the article. KTse, KTsi, and AM contributed to the writing of the article. All authors worked for the final version of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.