As care and antiretroviral treatment (ART) for people living with HIV/AIDS become widely available, the number of people accessing these resources also increases. Despite this exceptional progress, the estimated coverage in low- and middle-income countries is still less than half of all people who need treatment. In addition, treatment discontinuation and non-adherence are still concerns for ART programs. Governments and partner institutions have sought to implement a variety of interventions addressing the main reasons behind the low coverage of, discontinuation of, and non-adherence to ART. Food assistance is one of those interventions; increasing evidence suggests that this type of intervention has the potential to improve ART outcomes. However, to our knowledge, no study has estimated its costs in detail. The objective of this study was to assess the costs of a program providing food assistance to HIV/AIDS patients in Sofala province, Mozambique, in 2009.We performed a retrospective analysis of the costs of providing food assistance, based on financial and economic costs. We used the ingredients approach to estimate costs, which involved multiplying the total estimated quantities of goods and services actually employed in providing the intervention by their respective unit prices.In 2009, the cost of providing food assistance to HIV/AIDS patients was $2.27 million, with capital and recurrent costs accounting for 1% and 99% of total costs, respectively. Food made up the largest component, at 49% of total costs. At 24%, transport operating costs were the second largest item. The cost per patient served was $288 over 3 months.The food distribution program carries significant costs. To assess whether it provides value for money, the present study results should be interpreted in conjunction with the program’s impact, and in comparison with other programs that aim to improve adherence to ART. Our costing analysis revealed important management information, indicating that the program incurred relatively large overhead costs. This result raises questions regarding the efficiency of implementing this food distribution program. As care and antiretroviral treatment (ART) for people living with HIV/AIDS becomes widely available, the number of people accessing them also increases. At the end of 2010, 6.65 million people were receiving treatment in low- and middle-income countries, an increase of 27% from December 2009, since tGovernments and partner institutions such as the World Health Organization (WHO), the Joint United Nations Program on HIV/AIDS, the United Nations World Food Program (WFP), and major HIV/AIDS initiatives such as the US President’s Emergency Plan for AIDS Relief have sought to implement a variety of interventions addressing the main reasons behind the low coverage of, discontinuation of, and non-adherence to ART. Food assistance, which is aimed at improving the physical ability of patients to take ART and to render it more clinically effective, is one of those interventions-11. TherEstimating program costs is important for a number of reasons. First, costs could be used to assess allocative efficiency by determining whether a program provides value for money, for example, by comparing its costs and effects to those of other investments in health (e.g. food assistance versus tuberculosis control). Second, costs could be used to assess technical efficiency by determining whether a program provides value for money within the same domain, for instance, by comparing its costs and effects with those of alternative options for providing food assistance. Third, it could be used for management purposes, such as planning improvement by identifying larger-cost items and potentials for savings,21. In tWe evaluated the food assistance program as implemented by the United Nations WFP in collaboration with Health Alliance International (HAI) and the Ministry of Health. HAI is a non-governmental organization that initiated operations in Mozambique in 1987. Its mission is to support the development of policies that foster social and economic equity for all, with a focus on public-sector health systems and a progressive realization of the right to health. In 2003, in partnership with the government of Mozambique, HAI began to support the implementation and expansion of ART for HIV/AIDS patients. It is within this partnership that HAI also collaborates with the WFP in providing food assistance for HIV/AIDS patients in Sofala province.The WFP is responsible for the provision of food, health centers are responsible for the identification of eligible patients, and HAI is responsible for the distribution of food to patients. This intervention is provided to HIV/AIDS patients of all ages, including children. It is also provided to tuberculosis patients and to pregnant and breast-feeding women enrolled in prevention of mother-to-child transmission programs. The objective is to ensure patient nutritional recovery and treatment success.2,earning no income or less income than the monthly national minimum income, and many dependents. Patients were identified before or after they started ART. They were first identified by a social worker; patient eligibility was later confirmed by a clinician. After being identified, patients received an identification card that entitled them to collect food at the distribution site, usually a warehouse or health facility. They received food assistance once per month for a period of three months, after which they were reassessed to determine whether their nutritional status had improved. If improved, patients were discharged from the program; otherwise, the patients remained in the program for an additional 3–6 months. Food assistance consisted of 10 kg of soya, 5 kg of cowpeas, and 25 kg of maize.The criteria for inclusion in the food assistance program were body mass index below 18.5 kg/mThis study was approved by the Mozambican National Bioethics Committee.We undertook the costing analysis from a provider perspective: the costs incurred by the three organizations implementing the food assistance program . The cost calculations apply to all 13 districts in Sofala province over the implementation period of 2009.To estimate costs incurred by HAI and the health facilities, we used the ingredients approach by multiplying the total estimated quantities of goods and services actually employed in providing the intervention by their respective unit prices. This approach is useful because it makes it possible to later identify what is driving the total costs, and how to improve the use of available resources. We obtaTo estimate the costs incurred by WFP at the program level in Sofala province, we used a top-down approach based on the number of people reached by the intervention; we allocated transport and other program costs together (rather than separating transport and program) because we did not have information on the breakdown of costs at the national level by line item. We compared the total number of people served at the national level with the number of HIV/AIDS patients served in Sofala province, and used this proportion to estimate the costs at the province level. We distributed the program costs among categories according to the percentage of each category out of the total value of the national budget. For example, if staff costs corresponded to 6% of total value in the WFP budget allocated to Mozambique, we also allocated 6% of the food program costs in Sofala province for staff costs. We obtained details on costs incurred by the WFP in their budget allocated to Mozambique.To determine the costs of the intervention, we interviewed people in various job categories and visited five places where the intervention was actually implemented. These places were selected based on convenience of access. We divided the costs identified according to the activities performed in two phases of program implementation of the program: the start-up and post start-up phases. For the start-up phase (the time between the decision to implement an intervention and starting its delivery to the first beneficiary), we only included data on the costs of initial training . For the post start-up phase (the period of implementation), we included costs based on three main activities: planning and monitoring, patient identification, and food distribution.For planning and monitoring, we only included staff costs. For the health facilities, we based our estimates on expenditure data and included staff costs at the provincial level . For HAI (the organization responsible for food distribution), we included staff costs at the national and provincial levels  and at the district level (multidisciplinary team manager and administrative assistant). To assess staff time spent in these activities, we interviewed four administrative assistants, one nutritional assistant, the program manager, the program coordinator, and three multidisciplinary team managers. The nutritional assistant provided us with estimates of time spent by the health director and the chief medical doctor.Similarly, for patient identification, we only included staff costs incurred at the district level by health facilities, since these were responsible for the identification of patients eligible to receive food assistance. We included the costs of 13 health facilities, one health facility per district. To assess staff time spent at the health facilities, we interviewed five social workers, who also provided estimates of time spent by medical doctors.Regarding food distribution, we only included the costs incurred by HAI, since it was responsible for food distribution. We included the costs of transport, materials and supplies, equipment, training, and staff at the district level Table . To asseFor WFP, the organization that provided the food, we included the costs of staff, transport, and equipment. We obtained details about the price and quantities of food from an interview with the head of the program unit in Sofala province.We measured financial costs at the time the expenditure was incurred. We included staff, food, materials and supplies, and transport operational costs as recurrent costs Table . ForeignWe calculated economic costs by annuitizing capital goods on the basis of their useful life at a discount rate of 3%, as recommended by most guidelines. We inclWe calculated the cost per patient served by dividing the total costs of providing food assistance by the number of patients served . We implemented a sensitivity analysis to determine whether the results were sensitive to discount rate, staff time use, equipment prices, and the overhead costs of HAI and the health facilities.For the year 2009, the costs of providing food assistance to HIV/AIDS patients was $2,271,656 , followed by HAI (44.9%) and health facilities  to ensure the minimum acceptable consumption levels of people who are already in difficulty, (ii) to prevent people who are susceptible to adverse events and shocks from becoming more vulnerable (by stopping them from having to sell their assets), (iii) to promote people’s ability to become less vulnerable in the future (by helping them to build assets and achieve stronger livelihoods), and (iv) to promote social justice through promoting the rights and empowerment of the poor and vulnerable.Our findings reveal important information for program budgeting and for the identification of potential cost savings. Notably, the costs of food distribution were as high as the costs of the food itself, both accounting for approximately half of the total costs. If the food program were scaled up, little economies of scale should be expected, as these costs are all variable.Several of the limitations of the present study may be addressable by future investigations. First, we only included data on the costs of training for the start-up phase, which may have underestimated the true costs incurred during this phase. Second, we did not include the overhead costs of the HAI and the health facilities in our analysis, but these costs may be significant. Third, we did not include the time and travel costs that patients incur to receive food assistance. Insights into these patient costs would allow program decisions regarding the optimal location for food distribution. Fourth, we used a top-down approach to estimate costs incurred by the WFP. This allocation of program costs on the basis of the number of people reached may have contributed to the high share of the WFP in total costs. Future research could attempt to address this limitation by measuring all resource use via a more detailed approach, such as the ingredients approach, in order to improve cost estimation. Fifth, we only analyzed the costs of the food and of program implementation, and did not consider the quality of the implementation. Future research could address this issue by including an assessment of the process of beneficiary targeting. Insights into these issues would allow program managers to better understand whether the program was delivered as expected and perhaps aid in explaining why a program is or not effective.Despite calls for more economic analysis of interventions to improve treatment outcomes,28-31, tThe food distribution program carries significant costs. To assess whether it provides value for money, the present study results should be interpreted in conjunction with the program’s impact, and in comparison with other programs that aim to improve adherence to ART. Our costing analysis revealed important management information, in the sense that the program incurred relatively large overhead costs. This observation raises questions regarding the efficiency of its implementation.The authors declare that there are no competing interests.Both authors contributed to the design of the study. MP collected and analyzed data and drafted the manuscript. RB contributed advice on all aspects of study implementation and a critical revision of data analysis, interpretation of the results, and the manuscript. Both authors approved the manuscript.


The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction.Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from  High resolution images av3JPcdh15 (av3J), av6JPcdh15 (av6J) and v2JCdh23 (v2J) mice. The av3J mice carry a presumptive null allele of Pcdh15, and the av6J mice carry an in-frame deletion predicted to remove most of the 9th cadherin repeat from the ectodomain of PCDH15  in the +/av6J heterozygotes versus 34.9±2.8 (n = 6) in the av6J/av6J mutants, and in the basal coils the mean grey scale levels were 87.4±4.6 (n = 11) in the +/av6J heterozygotes versus 69.6±7.1 (n = 6) in the av6J/av6J mutants. The differences in grey scale levels between heterozygotes and mutants were not statistically significant (ANOVA plus Tukey test). In contrast, loading of FM1-43 dye was not detected at all in hair cells from either the apical or the basal coils of cochlear cultures prepared from av3J/av3J  all responded with large transducer currents when the hair bundles were deflected in the positive direction (towards the kinocilium) using saturating force stimuli (+/av6J OHCs (range −700 to −1230 pA), −540 pA for the +/av3J OHCs (range −370 to −850 pA) and −300 pA for the +/v2J OHCs (range −240 to −350 pA), showing considerable variability as reported before for rat OHCs 2+ ions through the transducer channels To determine whether the mechanotransducer channels could be activated by hair bundle displacements in the mutants, currents were measured in the whole cell mode whilst stimulating the hair bundles with a fluid jet . Heteroz stimuli . At −104av6J/av6J mutants, transducer currents could be elicited over the range of membrane potentials tested , larger transducer currents in response to stimuli directed away from the kinocilium, also at negative membrane potentials. This suggests that large transducer currents can be elicited, but they are not activated at rest and large forces are required to evoke them. The homozygous v2J/v2J mutants had transducer currents at all membrane potentials (+/v2J and three v2J/v2J OHCs) show the relatively small transducer currents observed at negative potentials for the homozygous mutant OHCs, which were 81% smaller than those of the heterozygous controls at −104 mV. Note that at positive potentials the currents are actually larger in the homozygotes, most likely because the heterozygous control currents happened to be rather small in this sample of two OHCs from a single mouse.In the s tested . The curs tested , averagetentials , where ctentials , but agaav3J/av3J and v2J/v2J OHCs is that they might not be transducer currents but some artefact, for example, of modulating the seal resistance between the patch pipette and the cell membrane. The currents in one of these mutants, v2J/v2J, were therefore investigated further, using mechanical step stimuli. At a hyperpolarized holding potential of −84 mV a considerably larger driver voltage to the fluid jet, and hence force on the hair bundle, was needed to elicit currents from the mutant v2J/v2J OHC than was required for the control +/v2J OHC  in the av6J mouse at P9, and in both the av3J and v2J mice at P4. Utricular bundles were examined by transmission electron microscopy (TEM) in the av6J and av3J mice at P15 and P26, and in the v2J mice at P11. In scanning electron micrographs, links were categorized as tip links if they emanated from the tip of a stereocilium in any of the first 3–4 tallest rows and attached to the side of a taller stereocilium in the next row. In immature bundles , such tip links could be directed to either or both of the two nearest taller stereocilia Electron microscopy was used to assess the status of tip links in +/av6J  the homozygote had fewer occupied sites (30%) compared with the heterozygote (46%), fewer unforked links (23% vs 47%) and fewer stumps (5% vs 9%) but more ambiguous links (65% vs 38%). Since most of the hair bundles had some abnormality in row structure, the number of possible sites was smaller in the homozygote than the heterozygote.Tip-link like links were present in both +/av6J  and av6Jls at P9 . These tls at P9  similar ls at P9  and latels at P9 . To dete+/av3J  of potential sites were occupied by a tip link, whereas only 46% (14/30) were found to have tip links in the av6J/av6J mutant. For the +/av3J mouse 80% (32/40) sites were occupied whilst 0% (0/43) was occupied in the av3J/av3J mutant. For the +/v2J mouse 43% (23/53) sites were occupied whereas 4.3% (2/46) were occupied in the v2J/v2J mice. The top of the shorter stereocilium at tip-link sites was often found to be wedge-shaped in the +/av6J and av6J/av6J mice, as well as in the +/av3J and the +/v2J mice  show that CDH23 and PCDH15 are required for the formation and/or maintenance of a substantial complement of normal tip links, 2) reveal that transducer currents are abnormal in mice with mutations in the genes encoding these proteins, and 3) provide further evidence that ototoxic aminoglycoside antibiotics enter hair cells via their mechanotransducer channels. The study therefore adds to a growing body of data v2J and the av3J mutants, and less so in the av6J mutants. The presumed loss-of-function mutations in Cdh23 (v2J) and Pcdh15 (av3J) therefore cause a similar phenotype, and the in-frame deletion of Pcdh15 (av6J) has an effect that is, at least initially, less severe than that observed in either of the presumed nulls. Although a previous study v/v and v2J/v2J mice, we were unable to find links with SEM that could be clearly defined as tip links in the v2J/v2J mice. The tip-link like links that are observed in the v2J/v2J mice are short and hard to distinguish from the abundant transient links present at these early stages. Tip links were, however, observed in the cochlea of the av6J/av6J mice. Whilst their numbers were somewhat reduced, they were clearly present. Likewise, in the relatively mature utricle where the effects of the mutations on hair bundle structure are less severe, we were able to find a number of clear examples of tip links in the av6J/av6J mutants with TEM, but not in either the av3J/av3J or the v2J/v2J mice. The only two links that were considered to be tip links in the utricles of v2J/v2J mice were not clear-cut, unambiguous examples of tip links. As in the cochleae, fewer potential tip link sites were occupied by a defined tip link in the av6J/av6J mutants than in the heterozygous +/av6J controls. These results therefore provide genetic evidence that PCDH15 and CDH23 are both required for the formation and/or maintenance of tip links, and that tip links can form in mutants in which a large part of the 9th cadherin repeat in the ectodomain of PCDH15 is deleted.SEM and phalloidin-stained preparations of the early postnatal cochlea revealed that hair-bundle structure was severely disrupted in both the av6J cuts out key structural elements from one of the cadherin repeats in PCDH15, several lines of evidence indicate that functional tip links are formed in the av6J/av6J mutants. The hair cells load with FM1-43, a dye that is known to enter hair cells via their transducer channels av6J deletion generates an entirely non-functional molecule that cannot interact with CDH23, one could argue that PCDH15 is not required for either tip link formation or transduction, despite accumulating evidence (including the data presented above) suggesting otherwise. An alternative and perhaps more parsimonious hypothesis would be that the deleted form of PCDH15 can interact with CDH23 and that tip links do form but are rather unstable. The reduction in the number of tip links observed in the av6J/av6J mutants, the reduced staining seen with antibody HL5614 to the PCDH15 ectodomain, the decrease in FM1-43 dye loading and the smaller currents recorded in response to hair bundle displacement all suggest that the latter hypothesis is correct. The behavioural and audiological deficits seen in the young av6J/av6J mouse av6J/av6J mouse that cuts out more of the cadherin repeats in a ‘structurally-feasible’ fashion , the available evidence indicates that 15 of the 17 predicted transmembrane splice variants expressed in the early postnatal cochlea include exon 22 Pcdh15 excluding exon 22 do not exist the early postnatal cochlea.Although the deletion in av6J/av6J mouse. The resting transducer conductance was reduced and the evoked currents were, on average, ∼50% lower in amplitude. These findings are, respectively, in accordance with the reduction in FM1-43 dye loading and the decreased number of unambiguous tip links seen in the av6J/av6J mouse. In the homozygous av3J/av3J and v2J/v2J hair cells there was no evidence for a resting current, as also predicted from the FM1-43 experiments in which no dye loading was observed. Quite unexpectedly, however, we found evidence for transduction in both of these presumed null mutants. In the homozygous av3J/av3J and v2J/v2J OHCs, small transducer currents were observed at negative holding potentials, but in response to force stimuli directed away from the kinocilium, whilst larger transducer currents could be elicited at positive potentials. In contrast to the transducer currents of the homozygous av6J/av6J OHCs, which appeared to be a scaled-down version of the currents in their heterozygous +/av6J counterparts, the v2J/v2J hair bundles required considerable displacement for the transducer channels to open and the polarity of the responses was abnormal, with bundle movements in the negative direction eliciting the largest transducer currents in all but one of the five OHCs tested with step stimuli. Three v2J/v2J OHCs tested with force steps responded with transducer currents to both negative (largest) and positive  stimuli. This suggests a degree of randomization of the directional sensitivity of the transducer channels, as previously reported for Vlgr1 knockout mice that lack ankle links and have disorganised hair bundles In response to hair-bundle displacements evoked by a fluid jet, transducer currents could be recorded from the hair cells of the homozygous av3J/av3J and v2J/v2J mutants in response to bundle displacements, together with our morphological observations, suggest that some form of transduction may occur in the absence of bona fide tip links . Alternative channel gating mechanisms, based on a kinematic analysis of stereociliary shearing and the localisation of a putative mechanotransducer transducer channel to the contact region that lies between stereocilia just below the tip link, have been suggested in the past av3J/av3J and v2J/v2J mutants, it is hard to see how these would mediate excitatory responses to deflections that are normally inhibitory . A gating mechanism for the mechanotransducer channels that lacks the directional sensitivity provided by an oblique tip link would be an alternative hypothesis, such as that proposed for inner (but not outer) hair cells in MYO15A mutant mice av3J and v2J mutants described above is that loss of normal tip links results in the channels being mislocalised to sites where they could be gated by the lateral links. This would imply that a residual gating element is present that enables the channels to be activated by stresses imposed on the membrane even in the absence of the normal link.The transducer currents recorded from v2J/v2J mouse defective in CDH23 and the av3J/av3J mouse defective in PCDH15 described here and those of the shaker1 mouse that is defective in MYO7A v2J/v2J mutant OHCs we identified with step stimuli are similar to the currents we observed previously in shaker 1 mutant mice av3J/av3J mutant mice do not accumulate [3H]-gentamicin and are insensitive to aminoglycosides. The cytoplasmic tail of MYO7A interacts directly with CDH23 Finally it can be noted that there are additional similarities between the hair cells of the To conclude, our results together with those of previous studies provide genetic evidence for the concerted involvement of PCDH15, CDH23 and MYO7A in the formation and regulation of the tip-link complex in mechanosensory hair cells. They also raise the intriguing possibility that the mechanotransducer channels may, under certain circumstances, be gated via mechanisms that do not involve the classic, oblique tip link.The use of mice at Case Western Reserve University (CWRU) was approved under protocol number 2010-0074 (entitled ‘Characterization of mouse models of deafness’) by the Institutional Animal Care and Use Committee (IACUC). The main objective of protocol 2010-0074 is to use various tools and techniques to understand the molecular basis of hearing and deafness using mouse models. At the University of Sussex, all animal procedures were performed under a UK Home Office Project licence  and with the approval of The University of Sussex Ethical Review Committee.Pcdh15, two alleles of Ames waltzer (av) were used: av6JPcdh15av3JPcdh15av mice was compared to that obtained from hair cells from mice with a mutation in Cdh23, the waltzer v2J allele. Mice homozygous for the av6J allele (av6J/av6J) were crossed to mice heterozygous for av6J allele (+/av6J) and offspring were genotyped using a PCR-based approach +/av3J) or homozygous (av3J/av3J) for the av3J allele of Pcdh15, and mice that were heterozygous (+/v2J) or homozygous (v2J/v2J) for the v2J allele of Cdh23. Offspring from the av3J and av6J lines were genotyped as described previously by Pawlowski et al and Zheng et al respectively v2J line were genotyped using a scheme that takes advantage of the point mutation that results in the loss of HpyCH4 III restriction site in the target sequence. Genomic DNA (∼500 ng/reaction) was amplified under standard reaction conditions using Platinum Taq DNA polymerase . The reaction mix was incubated at 94°C for 120 s followed by 35 cycles of 94°C for 30 s, 55°C for 20 seconds and 72°C for 30 s, 72°C for 45 s. Primer pairs and product size are as follows: Primer KA1063 – 5′CGA GAC CAA GAC CAG CTA CC 3′ and KA1064 – 5′ACC AGA CTG ACT GGC TTT CA, 412 bp. The PCR product was digested with HpyCH4 III  according to manufacturer's protocol followed by heat inactivation at 80°C for 15 minutes. The digested sample was resolved on a 3% agarose gel. Three bands  were observed with wild-type allele and two bands (372 and 40 bp) were observed for the v2J allele.To study changes associated with mutations in av3J, av6J and v2J mice at 0–3 days of age as described previously Cochlear organ cultures were prepared from +/av6J and av6J/av6J hair cells , +/av6J (n = 11), av3J/av3J (n = 4), av6J/av6J (n = 6). Similarly, apical- and basal-coil explants from +/v2J (n = 5) and v2J/v2J (n = 6) were cultured for FM1-43 labeling.FM1-43 dye loading experiments were performed as described previously ells see . Images 3H]-gentamicin labelling was performed as described previously 3H]-gentamicin  for 2 h at 37°C. Cultures were then placed on ice, washed three times with 4 ml of ice cold HBHBSS over a 10 minute period and fixed in cold 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2 for 1 h. Following glutaraldehyde fixation, cultures were washed thrice in 0.1 M cacodylate buffer, fixed for 1 h with 1% osmium tetroxide, washed in buffer, dehydrated with ethanol and embedded in Epon 812 resin . One-micrometer thick sections of the apical and basal-coil cochlear cultures were cut from the plastic blocks with glass knives, mounted on glass slides and either stained with Toluidine blue or coated with Ilford L4 Nuclear Research Emulsion  for autoradiography. Emulsion-coated slides were exposed in light-tight boxes in the presence of dessicant at 4°C for 1 to 3 weeks, and developed in Ilford Phenisol (diluted 1+4 with H2O) for 4 min at 18°C. Developed autoradiographs were washed, fixed and dried, mounted under glass coverslips in Histomount, and photographed with an Axioplan 2 light microscope using phase contrast optics.[To test for sensitivity to aminoglycoside exposure, coverslips with adherent cochlear cultures were placed in 35 mm plastic petri dishes, washed once with 3 ml of HBHBSS and then incubated in 3 ml of HBHBSS or 3 ml of HBHBS containing 1 mM neomycin or 1 mM gentamicin at 37°C for 2–4 hours. Following a brief wash, cultures were fixed as described above for grain density autoradiography. Effects of aminoglycosides were evaluated by light microscopy using 1 micron-thick, Toluidine blue-stained sections of basal-coil cultures.av3J mice (P2 plus two days in vitro) and v2J mice (P1 plus 2 or P0 plus 3 days in vitro), and from the cochleae of av6J mice that were acutely dissected at P4 and P5. All experiments were at room temperature (22–25°C). Extracellular solution was continuously bath-applied at a rate of 6 ml/h. It contained (in mM): 135 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO2, 2 Na-pyruvate, 5.6 D-glucose, 10 HEPES, with amino acids and vitamins for Eagle's minimum essential medium (MEM) added from concentrates . The pH was adjusted to 7.5 and the osmolality was about 308 mmol kg−1. The organs of Corti were observed with an upright microscope  with Nomarski optics.Mechano-electrical transducer currents from apical-coil outer hair cells were studied in organotypic cochlear cultures of 2, 1 EGTA-CsOH, 2.5 Na2ATP, 5 HEPES; pH adjusted to 7.3 with 1 M CsOH. Data were filtered at 2.5–3.0 kHz, sampled at 5 kHz and stored on computer for off-line analysis using Origin software. Membrane capacitance (Cm) was 5.9±0.3 pF and series resistance after electronic compensation of up to 70% (Rs) was 2.5±0.3 MΩ, resulting in voltage-clamp time constants of 15±2 µs (n = 21). Membrane potentials were corrected for a −4 mV liquid junction potential between pipette and bath solutions. No correction was made for the voltage drop across this series resistance, which was at most 2 mV at extreme potentials. Voltage clamp protocols are referred to a holding potential of −84 mV. Mean values are quoted ± s.e.m. in text and figures.Transducer currents were elicited using fluid-jet stimulation  and recorded under whole-cell voltage clamp  as previously described av6J mice, cochlear coils were stained only with Texas Red conjugated phalloidin.Cochleae or cochlear cultures were fixed by immersion in 3.7% formaldehyde in 0.1 M sodium phosphate buffer for 1 hour at RT, and washed 3 times in PBS. After removal of the cartilaginous capsule and the lateral wall, cochlear coils were preblocked and permeabilised in TBS containing 10% heat inactivated horse serum and 0.1% TX-100 for 1 hour, and incubated overnight in the same solution containing a 1∶100 dilution of each of the following affinity purified rabbit antibodies directed against PCDH15 : PB303, 464-2B and PB375 recognising the cytoplasmic domains of the CD1, CD2 and CD3 isoforms classes of PCDH15 respectively, and HL5614 directed against an N-terminal ectodomain sequence common to all classes. Antibody HL5614 was used in the presence of 2 mM EDTA. Samples were washed 3× with TBS, and stained with FITC conjugated swine anti-rabbit Ig (1∶100 dilution) and rhodamine conjugated phalloidin (1∶1000 dilution) for 2 h, washed in TBS, mounted in Vectashield and viewed with a Zeiss LSM 510 confocal microscope using a 100× oil immersion lens NA1.4. To examine hair bundle morphology in av3J mice at P15 and P26 and av6J mice at P15 were dissected in HBHBSS and fixed immediately in glutaraldehyde followed by osmium tetroxide and embedded in resin as described above for grain density autoradiography. For v2J mice the utricular maculae were dissected in 70% ethanol from labyrinths that had been immersion fixed in glutaraldehyde followed by osmium. Thin (∼90 nm thick) sections were cut with a diamond knife, mounted on copper grids, double stained with uranyl acetate and lead citrate +/av6J, 4 av6J/av6J, 6 +/av3J, 7 av3J/av3J, 4 +/v2J and 2 v2J/v2J mice were examined by transmission electron microscopy.Utricles from av6J mice at P9, v2J at P4 and av3J mice at P3 were fixed by intra-labyrinthine perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 2 mM CaCl2 (pH 7.4) and then immersed in the fixative for 2 h. They were stored in 1/10th concentration fixative  diluted with buffer at 4°C until further processing, subsequently washed in buffer, dissected to reveal the organ of Corti, and post-fixed with cacodylate-buffered 1% OsO4 for 1 h. After washing in cacodylate buffer, samples were impregnated with osmium using the osmium-thiocarbohydride (OTOTO) method; for details see 2 using a Polaron critical point dryer. Cochleae were affixed to specimen stubs using silver conducting paint  and examined in a Hitachi S4500 field emission SEM operated at 5 kV. Specimens from the cochleae of 4 +/av6J, 2 av6J/av6J, 2 +/v2J, 2 v2J/v2J, 2 +/av3J and 2 av3J/av3J mice were examined by scanning electron microscopy.Cochleae from heterozygous and homozygous


Streptococcus pneumoniae has the ability to use the carbon- and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previously shown to contribute to pneumococcal virulence. To get a broader understanding of the response of S. pneumoniae to cellobiose, we compared the pneumococcal transcriptome during growth on glucose as the main carbon source to that with cellobiose as the main carbon source. The expression of various carbon metabolic genes was altered, including a PTS operon (which we here denote as the bgu operon) that has high similarity with the cel locus. In contrast to the cel locus, the bgu operon is conserved in all sequenced strains of S. pneumoniae, indicating an important physiological function in the lifestyle of pneumococci. We next characterized the transcriptional regulation of the bgu operon in more detail. Its expression was increased in the presence of cellobiose, and decreased in the presence of glucose. A novel GntR-type transcriptional regulator (which we here denote as BguR) was shown to act as a transcriptional repressor of the bgu operon and its repressive effect was relieved in the presence of cellobiose. BguR-dependent repression was demonstrated to be mediated by a 20-bp DNA operator site (5′-AAAAATGTCTAGACAAATTT-3′) present in PbguA, as verified by promoter truncation experiments. In conclusion, we have identified a new cellobiose-responsive PTS operon, together with its transcriptional regulator in S. pneumoniae.The human pathogen  Streptococcus pneumoniae is a Gram-positive bacterial pathogen in humans that has the ability to colonize the nasopharyngeal cavity of the nose S. pneumoniae must have the ability to adapt to fluctuating levels of nutrients S. pneumoniae is able to use an exceptionally broad spectrum of different carbon sources like cellobiose, raffinose, sucrose, galactose, maltose and others S. pneumoniae as an energy source alternative to a preferred sugar like glucose S. pneumoniae, i.e. the Cel system Previous studies have shown that cel locus) S. pneumoniae strain R6 to grow on cellobiose as the sole energy source. The cel loci of strains R6/D39 consist of seven genes and they are transcribed into two transcriptional units cel locus encode a phospho-β-glucosidase (celA), a DNA-binding transcriptional activator (celR), cellobiose-specific PTS IIBAC components  and two proteins with unknown functions. CelR was found to be responsible for activation of the cel locus in Streptococcus mutans in the presence of cellobiose cel locus in S. pneumoniaecel locus is not conserved in all pneumococcal strains, as it is absent in 50% of the sequenced S. pneumoniae genomes available on the KEGG website, including major multi-drug resistant pneumococcal strains like 19F and 23F. Based on this observation, we hypothesized that S. pneumoniae might possess alternative systems that it employs to use β-glucosides like cellobiose.The Cel system , PbguA-5.4 (first 6 bases of the BguR operator site deleted) and PbguA-5.5 . This resulted in plasmids pSS302-04. These constructs were introduced into D39 wild-type as described above, resulting in strains SS305-07. All plasmid constructs were checked by sequencing.The following promoter subclones of PSpecific β-galactosidase assays were performed as described before S. pneumoniae, the D39 wild-type strain was grown in 3 biological replicates in CM17 (0.5% Cellobiose+M17) and compared to the strain grown in GM17 (0.5% Glucose+M17) medium. Cells were harvested for RNA isolation at two time points in CM17 (CT-1 and CT2) and GM17 (GT-1 and GT-2) medium  were grown in 3 biological replicates in GM17 (0.5% Glucose+M17) medium. These cultures were harvested at the mid-exponential phase of growth at an OD600 of 0.25. All other procedures regarding the DNA microarray experiments , hybridization and scanning) were performed as described before For transcriptome analyses of dium see . To analDNA microarray data were analyzed as described before www.ncbi.nlm.nih.gov/BLAST/) was used for blasting genes among different genomes, while the Kyoto Encyclopedia of Genes and Genomes (www.genome.jp/kegg/) database was used to analyze different genomes of S. pneumoniae  for the presence of the cel gene cluster and bgu operon. The in-house developed software Genome 2D (http://server.molgenrug.nl/) was used to analyze the whole genome of S. pneumoniae D39 for the presence of BguR operator sites, allowing 4 mismatches.The NCBI web site  that encodes a putative multiple sugar transport system was highly upregulated as well. Cellobiose metabolism has previously been shown to be carried out by a cellobiose-specific gene cluster (cel locus) in streptococci, including S. pneumoniaecel locus, specifically in the presence of cellobiose cel locus was also among the highly upregulated genes at both time points. This suggests that the conditions applied for the transcriptome analyses are indeed appropriate to find cellobiose-responsive genes. The expression of an operon  encoding a glycosyl hydrolase protein and the PTS system IICBA components was also highly upregulated at both time points of growth. Interestingly, blast searches showed that this operon has high similarity with the cel locus that was previously identified to be involved in cellobiose metabolism bgu operon in the metabolism/utilization of cellobiose or similar carbon sources. Therefore, we decided to further investigate the regulation of this operon in the presence of cellobiose.The effects of cellobiose on the transcriptome of bgu locus spans the genes SPD1830-33 (SPD1830 (here named bguA) encodes a glycosyl hydrolase belonging to the BglB family, which has high similarity (30% identity) to celA of S. pneumoniae and other streptococci. Next, the downstream three genes , encode PTS system IICBA components that show high similarity (29–30% identity) with celDCB located in the cel locus of various streptococci, including S. pneumoniae. Upstream of bguA, a gene encoding a GntR family transcriptional factor (named bguR) is located. The presence of this transcription factor in the DNA region upstream of the bgu operon indicates that it may function as a transcriptional regulator of the bgu operon .The D1830-33 . SPD1830cel locus cel locus was found to be absent from 50% of the S. pneumoniae strains present in the KEGG database. We also analyzed all the strains of S. pneumoniae available on the KEGG website for the presence of the bgu operon. Notably, this operon is 100% conserved in all the sequenced strains of S. pneumoniae available on KEGG database. This suggests an important role of this operon in carbohydrate metabolism, probably of cellobiose or related/analogous compounds bgu operon.In a previous study, we have studied the role of CelR in the regulation of the bgu operon in the transcriptome analyses was due to a specific effect of cellobiose, we constructed an ectopic transcriptional lacZ-fusion to the bguA promoter in the D39 wild-type strain. This strain was grown in M17 medium supplemented with 0.5% of different carbon sources . The highest expression of PbguA was observed in the presence of cellobiose , which we named BguR. We hypothesized that this regulator could be involved in the observed regulation of the bgu operon. To investigate the role of BguR, the bguR gene was replaced by a spectinomycin-resistance marker by means of allelic replacement. To examine the effect of the bguR deletion on the transcriptome of S. pneumoniae, the transcriptome of the bguR mutant strain (SS300) was compared to that of the D39 wild-type strain grown in GM17 (0.5% glucose+M17) medium. GM17 medium was used, since low expression of the bgu operon was observed in the presence of glucose, which we hypothesized to represent a condition with maximal repression of the bgu operon by BguR. These transcriptome data revealed that the expression of 13 genes was significantly altered due the deletion of bguR (bguR was five-fold downregulated confirming the inactivation of the bguR gene in the bguR deletion strain (SS300). The most highly upregulated genes were the ones constituting the bgu operon (more than 20-fold), indicating a role/function of BguR as the repressor of the bgu operon. Another strong effect caused by deletion of bguR was the upregulation of an operon involved in maltose/maltodextrin metabolism bguR brings about only a modest change in the transcriptome of S. pneumoniae D39, and furthermore imply a role of BguR in repressing the expression of the bgu operon.Adjacent to the  of bguR . The expbgu operon that was upregulated in the bguR mutant strain (SS300), we introduced the PbguA-lacZ into D39 wild-type and the bguR mutant. In GM17 (0.5% Glucose+M17) medium specific β-galactosidase activity was highly increased in the bguR mutant as compared to D39 wild-type  was found in the PbguA promoter bgu operon in S. pneumoniae is independent of CcpA.CcpA is considered a master transcriptional regulator in the control of carbohydrate utilization and metabolism genes in Gram-positive bacteria including  glucose . In addiS. mutans, regulation of the cel locus by CelR requires the phosphorylation of CelR by one of the PTS components, namely CelD bgu operon via an effect on BguR activity, we measured specific β-galactosidase activity of PbguA-lacZ in a bguDBC mutant strain. No difference in expression of PbguA was observed in the bguDBC mutant as compared to the wild-type D39 strain, when cells were grown in M17 medium with cellobiose or glucose  relieved the repressive action of BguR on PbguA, suggesting the presence of a putative BguR operator in this deleted region of the promoter. Further bioinformatics analysis of this area revealed the presence of a 20-bp palindromic region (5′-AAAAATGTCTAGACAAATTT-3′) that is overlapping with the -35 site and that might act as the BguR operator site. Deletion of half of this predicted operator site (PbguA-5.5) led to high expression of PbguA in CM17 (0.5% Cellobiose+M17) and GM17 (0.5% Glucose+M17) medium in the wild-type (bguA was truncated only a few base pairs upstream of the predicted operator site (PbguA-5.3), expression was similar to that of the full-length promoter  celR-bguDBC in the presence of 0.5% cellobiose and 0.5% glucose with that of D39 wild-type  cel locus is not conserved in all the sequenced strains of S. pneumoniae that are available on the KEGG website. This might indicate that S. pneumoniae harbors other ways of cellobiose utilization. To be able to more thoroughly understand the effect of cellobiose on S. pneumoniae, we applied transcriptome profiling and various ways to study transcriptional gene regulation. The data show that, besides the cel locus, expression of a second operon (bgu) is responsive to cellobiose as well, which we show to be mediated by the transcriptional regulator BguR. The fact that the bgu operon is 100% conserved in different pneumococcal strains suggests that it has an important physiological function in the life style of S. pneumoniae. Indeed, like celR, bguD was also found in one of the STM (Signature-tagged mutagenesis) studies, where it was implicated in lung infection In many bacteria, including S. mutans, it has been shown that CelR has two PRD (PTS regulation domain) domains and phosphorylation (by glucose and cellobiose PTSs) at H226, H332 and H576 is required for the activation of CelR in the presence of cellobiose, while phosphorylation of H284 and H391 leads to inhibition of the CelR-dependent activation in the presence of glucose S. pneumoniaeThe phosphoenolpyruvate-dependent (PEP) phosphotransferase system (PTS) is a major carbohydrate uptake system in bacteria, which not only phosphorylates different carbohydrates during uptake but also plays a major role in genetic regulation of metabolic activities bgu PTS genes on the cellobiose- and glucose-dependent regulation of PbguA via BguR. Therefore, it remains to be determined how the signal of the carbohydrate source is transferred to BguR. In addition, no effect of CcpA on the regulation of the bgu operon could be detected, in accordance with previous studies bgu locus seems to be governed by a single transcriptional regulator, BguR, possible via direct binding of glucose and cellobiose to its C-terminal UTRA (UbiC transcription regulator-associated) domain and the regulation of the bgu locus is independent of the Cel system.In the BguR amino acid sequence no similar regulatory domains could be identified. In agreement with this, we could not find an effect of deletion of the cel locus and the CelR regulatory site of S. pneumoniae were found to be highly conserved in other streptococci, although not all pneumococcal strains contain this operon bgu operon in all the strains of S. pneumoniae that are available on the KEGG website. However, no similar bgu operon organization could be found in other streptococcal species using BLAST searches. Thus, the bgu operon seems to be specific for S. pneumoniae.As reported in our previous study, the Bacillus subtilisEscherichia coliStreptomyces coelicolorgmuBACDREFG operon, encoding glucomannan utilization genes bgu operon on growth on cellobiose, it could be involved in the utilization of other glucomannan degradation products such as 4-O-β-D-mannopyranosyl-D-mannopyranose , and 4-O-β-D-glucopyranosyl-D-mannopyranose. Intestinal anaerobic bacteria encounter glucomannan and are able to degrade and ferment it S. pneumoniae as well, by uptake from the intestine.Blast searches with BguR revealed high similarity of this protein with the previously characterized GntR-type transcriptional regulator GmuR in bgc operon which consists of 5 genes including genes encoding PTS subunits IIB (BgcE), IIA (BgcF), IIC (BgcI) and phospho-β-glucosidase (BgcA), and is found to be involved in the utilization of cellobiose and other β-glucosides  at low temperature in E. coliS. pneumoniaebgu operon, we cannot exclude a role of the bgu operon in the utilization of arbutin or analogous sugars that S. pneumoniae encounters in its natural environment.BgcR is a positive transcriptional regulator of a bguDBC) of the bgu operon has no effect on growth compared to the wild-type in the presence of cellobiose as the sole carbon source, whereas deletion of celR (impairing expression of the cel locus) does. Recent studies in S. pneumoniae R6 have shown that deletion of bguD did lead to an increased lag-phase on cellobiose and a slight effect on growth on amygdalin celD led to strong inhibition of growth of S. pneumoniae R6 on amygdalin, cellobiose, esculin and gentiobiose cel locus is primarily responsible for β-glucoside utilization in S. pneumoniae and the exact role of the bgu operon in the uptake and metabolism of β-glucosides remains to be determined.Deletion of the genes encoding the PTS components Click here for additional data file.


Farmington virus (FARV) is a rhabdovirus that was isolated from a wild bird during an outbreak of epizootic eastern equine encephalitis on a pheasant farm in Connecticut, USA.Rhabdoviridae.Analysis of the nearly complete genome sequence of the prototype CT AN 114 strain indicates that it encodes the five canonical rhabdovirus structural proteins  with alternative ORFs (> 180 nt) in the N and G genes. Phenotypic and genetic characterization of FARV has confirmed that it is a novel rhabdovirus and probably represents a new species within the family Rhabdoviridae.In sum, our analysis indicates that FARV represents a new species within the family  Rhabdoviridae is currently divided into nine approved genera ; however, a large number of animal and plant viruses that appear to be members of the family have not yet been characterized or approved as species () and transcription termination/polyadenylation (CUAA[A/U]U[G/C]UUUUUUGG) sequences. The five coding regions each contain a long ORF corresponding to the canonical rhabdovirus genomic organization 3′-N-P-M-G-L-5′  45.1 kDa protein that in pairwise alignments shares little sequence identity with the N proteins of other rhabdoviruses. BLASTp searches at NCBI and UniProt found no homology to any viral proteins. In addition, searches using the SMART software  revealedThe 951 nt FARV P ORF encodes a 35.8 kDa protein that shares no detectable homology with rhabdovirus P proteins, shares no significant identity with any characterized sequence when subjected to a BLASTp search, and no conserved domains were identified. However, like other rhabdovirus P proteins , the putative FARV P protein features a high proportion of charged residues (~32%), including various clusters in regions that are predominantly acidic or basic.The 441 nt FARV M ORF encodes a mildly acidic (pKa = 9.23) 16.4 kDa protein that again shares no significant identity with any other known proteins. However, the N-terminal domain does contain the late budding domain motif (L-domain) PPxY that is present in many other rhabdovirus M proteins and is involved in the recruitment of cellular proteins involved in the budding process -14.http://www.cbs.dtu.dk/services/NetNGlyc/), indicating the mature G protein has a predicted size of ~102 kDa, which is somewhat larger that the G proteins of most other known rhabdoviruses. There are 16 cysteine residues in the ectodomain, many of which appear to align with 12 highly conserved cysteine residues (CI – CXII) that occur commonly amongst animal rhabdovirus G proteins, forming 6 intramolecular disulphide bridges . Th. Th21]. doviridae. Due to Rhabdoviridae is a very diverse taxon, which includes viruses that infect a wide range of hosts. Due to the genetic diversity within the Rhabdoviridae, it has been difficult to identify evolutionary relationships between and within the most diverse groups. In addition, this has been complicated by viruses such as LBVaV which, although differing in morphology and genome organization, has similarities in their in the amino acid sequences of both the nucleoprotein (N protein) coding regions and RNA-dependent RNA polymerase (L protein) with those of rhabdoviruses [The family oviruses .Vesiculovirus as reported previously [vesiculovirus cannot be correlated with any sequence similarity. Our sequence analysis indicates little overall identity of FARV to the other known rhabdoviruses in most proteins, except for the polymerase. Immunoblot analysis employing the FARV MIAF that was used in CF tests indicated the major homologous reactions were with the G, N and P proteins. It is likely, therefore, that antigenic cross-reactions detected against vesiculoviruses in CF tests are due to common epitopes in one or other of these proteins. Indeed, pair-wise sequence alignments of the FARV N and P proteins with the corresponding vesiculovirus proteins did indicate several short stretches of amino acid sequence (8–9 amino acids) in with the sequence identity is high (not shown). Similar limited regions of high identity in the N protein have been reported previously to account for distant antigenic cross-reactions between the ephemeroviruses and lyssaviruses [When FARV was first isolated in 1969, it could not be identified and was deposited in the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) collection as an unknown virus. Later ultrastructural and serologic studies  indicateeviously . The re-aviruses .Rhabdoviridae is that the distance between the various groups is so large that any nucleotide alignment would not be any different from random, thus nucleotide alignments do not give a robust construction of the relationships between the different genera. Because of this, the global relationships across the Rhabdoviridae have previously been determined by using the RNA-dependent RNA polymerase domain of L gene protein sequence [Rhabdoviridae, there are insufficient sequences available in the public domain  to determine their deep evolutionary relationships.One of the major issues with using sequences as a means to determine relationships within the sequence . Even soRhabdoviridae but the evolutionary history and host adaptation processes that have led to genome segmentation and gene loss is less clear. An alternative tree in which the LBVaV was excluded, under the assumption that varicosaviruses may not be truly related to Rhabdoviridae, did not result in any different placement of FARV, nor to its bootstrap support.Genetically, FARV is also similar to the varicosavirus LBVaV. Varicosaviruses are negative-sense ssRNA viruses that have two linear RNA segments and infect plants (lettuce and tobacco) . As noteNucleorhabdovirus and Cytorhabdovirus along with LBVaV, unassigned genus Varicosavirus  was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch. As noted previously, CT AN 114 was isolated from an unidentified bird collected during an epidemiologic investigation of an outbreak of eastern equine encephalitis on a commercial pheasant farm in Farmington, CT in 1969. At the time of isolation, it could not be identified and was deposited in WRCEVA virus collection as an unknown. The virus strain used in our studies had been passaged 11 times by intracerebral inoculation of newborn mice and twice in Vero cells. The virus used for sequencing was prepared from a single plaque picked from a monolayer culture of Vero cells.Vero E6 cells (African green monkey kidney) were used for propagating the virus. The cells were originally obtained from the American Type Culture Collection  and were grown at 37°C in minimal essential medium (MEM) with Earle’s salts  supplemented with 5% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS) and 1% penicillin-streptomycin stock .Outbred ICR mice  were used to prepare antigens and antibodies for FARV and the other rhabdoviruses shown in Figure Antigens used in CF tests were prepared from infected newborn mouse brains extracted by the sucrose/acetone method . SpecifiCF tests were done according to a microtechnique described previously ,28, usin2 were added. The infected cell monolayers were washed in 0.1 M cacodylate buffer, scraped off, and processed further as a pellet. The pellets were post-fixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.3, for 1 h, washed with distilled water, and stained en bloc with 2% aqueous uranyl acetate for 20 min at 60°C. The pellets were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 . Ultrathin sections were cut on a Leica EM UC7 ultramicrotome , stained with lead citrate and examined in a Phillips 201 transmission electron microscope at 60 kV.For ultrastructural analysis, infected Vero cells were fixed for at least 1 h in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, to which 0.03% picric acid and 0.03% CaCl2 plastic tissue culture flasks and were infected with FARV or were mock infected. Virus inocula were aspirated following 1 h of absorption at 37°C, washed thrice to remove any unabsorbed virus, replaced with fresh medium (MEM) supplemented with 5% FBS and 1% penicillin/streptomycin, and incubated at 37°C. Four days post infection (p.i.), the cells were harvested into cell lysis buffer . The cell preparations were then treated according to the Biorad instructions for MiniPROTEAN TGX gel analysis of proteins. Briefly, 20 μl samples of loading buffer containing β-mercaptoethanol were added to 20 μl aliquots of the cell lysates. Samples were heated at 95°C for 10 min, and centrifuged for 1 min at 500 × g at 4°C. Proteins were separated under reducing conditions on MiniPROTEAN TGX 4-20% Tris-Glycine SDS-PAGE gels, and were transferred onto Hybond-P PVDF membrane (Amersham) with Transfer Buffer  according to the manufacturer’s western blotting transfer protocol. Membranes were processed by the WesternBreeze© Chromogenic Immunodetection system (Invitrogen), following the manufacturer’s protocol. The polyclonal MIAF raised to FARV was used at 1:5000 dilution.Vero monolayers were grown to 90% confluency in 25 cmDrosophila megalomaster sigmavirus (YP003126913); DObSV, Drosophila obscura sigmavirus (ACU65444); DURV, Durham virus (ADB88761); DUVV, Duvenhage virus (AFK93192); EVEX, eel virus European X (AFX58972); EBLV1, European bat lyssavirus 1 (YP001285392); FLAV, Flanders virus (AAN73288); HIRV, Hirame rhabdovirus (NP919035); IKOV, Ikoma lyssavirus (AFQ26098); IHNV, infectious hematopoietic necrosis virus (NP042681); IRKV, Irkut virus (ABV03823); ISFV, Isfahan virus (CAH17548); JURV, Jurona virus (AEG25349); KHUV, Khujand virus (ABV03824); KIMV, Kimberley virus (AFR67096); KOTV, kotonkan virus (YP006202628); LBV, Lagos bat virus (AFW16650); MARV, Maraba virus (AEI52253); MOKV, Mokola virus (YP142354); MOUV, Moussa virus (ACZ81407); NGAV, Ngaingan virus (YP003518294); OVRV, Oak Vale rhabdovirus (AEJ07650); OBOV, Obodhiang virus (YP0062000965); OZEV, Ozernoe virus (ACS70797); PRV, Perinet virus (AEG25355); PFRV, pike fry rhabdovirus (ACP28002); RABV, rabies virus (ABN11300); SHIBV, Shimoni bat virus (ADD84511); SCRV, Siniperca chuatsi rhabdovirus (YP802942); SHRV, snakehead virus (NP050585); SVCV, spring viraemia of carp virus (NP116748); TIBV, Tibrogargan virus (ADG86355); TUPV, tupaia virus (YP238534); VSAV, vesicular stomatitis Alagoas virus (ACB47443); VHSV, viral hemorrhagic septicemia virus (BAM29126); VSIV, vesicular stomatitis Indiana virus (NP041716); VSNJV, vesicular stomatitis New Jersey virus (AAA48442); WCBV, West Caucasian bat virus (ABV03821); and WONV, Wongabel virus (YP002333280). The ICTV has not yet recognized FARV as a member species in the family Rhabdoviridae. As a consequence, its abbreviation ought to be considered tentative and subject to approval.The genome sequence of FARV was determined in this study and assigned GenBank accession number KC602379. The GenBank accession numbers for the genome sequences of select rhabdoviruses used in the phylogenetic analyses are listed as follows: ARV, Ade laide River virus (AFR23540); ABLV, Australian bat lyssavirus (NP478343); ARAV, Aravan virus (ABV03822); BEFV, bovine ephemeral fever virus (NP065409); CHPV, Chandipura virus (AED98393); COCV, Cocal virus (ACB47438); CPV, Coastal Plains virus (ADG86364); DMelSV, RNA was extracted from virus stocks (infected Vero cells) using TRIzol LS (Invitrogen) and treated with DNase I . cDNA was generated using the Superscript II system (Invitrogen) employing random hexamers linked to an arbitrary 17-mer primer sequence , treatedGenomic termini were characterized with 5_- and 3_-RACE kits (Invitrogen). Virus-specific primers for FARV were: 5′ – GTC TTG AAG TCG TTT CCC AG, located 934 nt from the 3′ -genomic terminus for reverse transcription, 5′ – TCA GGT TCA TCA GCC ATT TC, located 618 nt from the 3′ – genomic terminus for first PCR with primer UAP (Invitrogen), and 5′ – ACC AGC CGA TGA TGT AAG C, located 556 nt from the 3′ – genomic terminus for second PCR with primer AUAP (Invitrogen). RNA for the second RACE was tailed with poly(A) polymerase (Ambion) and purified using RNeasy® Mini kit (Qiagen). cDNA synthesis was primed with oligo d(T) – adapter primer AP (Invitrogen), and first PCR used primer 5′ – AAC CGT TCC TTC ACT ACA TC, located 1,004 nt from the 3′– antigenomic terminus and primer UAP (Invitrogen), the second PCR used primer 5′ – TCG CTT ACC AGC ATT TTG AG, located 746 nt from the 3′ – antigenomic terminus and primer AUAP (Invitrogen). All primers were at 0.2 M final concentration. PCR products were purified with QIAquick PCR purification kits (Qiagen) and directly dideoxy-sequenced in both directions.The L protein sequence of FARV was compared with those of 38 other rhabdoviruses downloaded from GenBank. All protein sequences were aligned using MUSCLE  under deFARV: Farmington virus; CF: Complement fixation; CPE: Cytopathic effect; MIAF: Mouse immune ascitic fluid; CHPV: Chandipura virus; ISFV: Isfahan virus; MARV: Maraba virus; JURV: Jurona virus; LJV: LaJoya virus; ORF: Open reading frame; dpi: Days post-infection; LBVaV: Lettuce big-vein associated virus; WRCEVA: World reference center for emerging viruses and arboviruses; MEM: Minimal essential medium; FBS: Fetal bovine serum.The authors do hereby declare that they have no competing interests in this scientific work.GP, NS, NV, KdT, HG, RBT performed the laboratory experiments. APATdR performed the serologic assays. NFL performed the phylogenetic analyses; VP performed electron microscopy; PJW performed the genomic analyses. NV, PJW, NFL, WIL, CP, RBT contributed to final the manuscript preparation. All authors have read and approved the final manuscript.Lyssavirus (RABV- Rabies virus), Vesiculovirus (Vesicular stomatitis Indiana virus – VSIV), Ephemerovirus , Novirhabdovirus (Infectious hematopoietic necrosis virus – IHNV), Tibrovirus (Tibrogarban virus – TIBV), Sigmavirus (Drosophila melanogaster sigmavirus – DmSv), unassigned species Wongabel virus (WONV), Ngaingan virus (NGAV), Flanders virus (FLAV), Tupaia virus (TUPV) and Moussa virus (MOUV) and presently unclassified viruses Durham virus (DURV) and Niakha virus (NIAV). The predicted N-terminal signal peptides and C-terminal transmembrane domains are shaded in aqua; conserved cysteine residues are shaded in grey; and other relatively conserved residues are shaded in black. Cysteine residues (dark shading) are numbered according to the conserved patterns observed in all animal rhabdovirus G proteins.Amino acid sequence alignment of FARV and 13 other rhabdovirus G proteins representing approved genera Click here for file


A virus was isolated from mink showing clinical and pathological signs of enteritis in China. This virus, designated MEV/LN-10, was identified as mink enteritis virus (MEV) based on its cytopathic effect in the feline F81 cell line, the hemagglutination (HA) and hemagglutination inhibition (HI) assay, electron microscopy (EM) and animal infection experiments. The complete viral genome was cloned and sequenced. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and canine parvovirus (CPV). The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature. Our findings not only provide valuable evidence indicating that recombination is an important genetic mechanism contributing to the variation and evolution of MEV, but also that heterogeneous recombination can occur in the feline parvovirus subspecies. Mink enteritis virus (MEV) is an autonomous parvovirus causing an acute disease in mink. The disGenetic recombination is generally considered a key mechanism in virus evolution. Recently, a Japanese research group provided the first clear evidence of recombination both between different CPV antigenic types and between CPV and FPLV under field conditions,10. WithMustela vison) raised for fur production. Approximately 200 (50%) of the minks suffered from haemorrhagic diarrhea of which 25% (100) died within 7-10 days after the onset of clinical signs. The intestinal contents were collected from diarrheic minks, and the virus was isolated as described elsewhere[An MEV strain was recovered from clinically diseased minks. In August 2010, a severe outbreak of haemorrhagic enteritis occurred in a farm in Dandong City, Liaoning province, China consisting of approximately 400 minks , 10 pmol each of the corresponding primers, 0.5 μl LA Taq polymerase (2.5 U/ml)  and 18 μl distilled H2O. The following thermocycling conditions for the PCR amplification were applied: 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 s, primer annealing at 55°C for 45 s, and extension at 72°C for 2 min, with a final elongation at 72°C for 8 min. The amplicons were analyzed by electrophoresis on a 1% agarose gel in Tris acetate EDTA buffer.The full-length genome of the virus was sequenced according to methods described previously. Four ovPCR products of the correct size were gel purified and cloned into the pMD18-T vector . For each amplified genomic fragment, 3-5 positive recombinant plasmids were sequenced in both directions using the M13 primer  and a gene specific primer. The complete sequence determined in this study has been deposited with GenBank, under accession number HQ694567.Complete CPV, FPLV and MEV genomic sequences were obtained from GenBank Table. These CThe phylogenetic relationships between the different viral genomes were evaluated using MEGA version 5.0. Phylogenetic trees were constructed using the Maximum Likelihood method in MEGA5 and the Tamura-Nei model with 2000 bootstrap replicates to assess the confidence level of the branch pattern. Bootstrap values > 70% were considered to be significant. Nucleotide and amino acid sequences of MEV/LN-10 together with reference MEVs, and CPVs were analyzed using DNAMAN software.5.5 TCID50/ml of MEV/LN-10 presented clinical symptoms of disease consistent with parvovirus infection and the virus was subsequently recovered from the animals. All animal work and experimental procedures were approved by the Institutional Animal Care and Use Committee of Jinlin University, China.Typical parvovirus associated CPE appeared in the F81 cells inoculated with the isolated virus. The negatively stained virus particles extracted from the cell supernatant following centrifugation were approximately 20 nm in diameter when examined by EM, and displayed a typical MEV morphology (data not shown). The virus was highly resistant to heat (56°C for 30 min), 20% ether, and acid (pH 3.0), but was susceptible to the antiviral agent 5-iododeoxyuridine (5-IUDR) (data not shown). The HA assay indicated that the highest HA titer of the virus was 1:2,048 with porcine erythrocytes but < 1:2 with sheep, guinea pig, rat, rabbit, chicken and human erythrocytes. The ability to agglutinate pig erythrocytes was inhibited by specific antiserum in the HI assay. In artificial infection experiments of mink, all of the animals injected subcutaneously with 1 × 10Both the GARD and RDP3 programs detected a putative recombination breakpoint in the nucleotide sequence of MEV/LN-10. Apart from the recombinant FPLV strain XJ-1, there was no evidence supporting recombination events in the genome sequences of the remaining 23 strains of FPLV, MEV or CPV analyzed by GARD or RDP3. GARD identified nucleotide position 2,686 in the MEV/LN-10 as a putative breakpoint for recombination  to the corresponding part of strain CPV-447. The different breakpoint determined by MaxChi from that determined by GARD was due to MaxChi calculating the midpoint of nucleotide positions 2,680 and 2,752 as the putative breakpoint since no nucleotide difference was detected between these positions as determined from the alignments of MEV and CPV. The alignment was cut at two points  and separate phylogenetic trees for each of the three sections were constructed. Both the first  and the recombination  regions of the alignment clustered with the CPV clade while the third fragment  clustered with the FPLV/MEV clade. Analysis using other evolutionary models, such as the neighbor-joining method and Jukes–Cantor, produced trees with nearly identical topologies (data not shown).Moreover, a potential recombination event was detected using the MaxChi method in RDP3. MaxChi identified nucleotide position 2,707 as an ending breakpoint with a Bonferroni corrected A comparison of all the nucleotide variations and the deduced amino acid sequences of the NS1 and VP1 VP2) proteins of the MEV/LN-10 strain with those of reference viruses MEVB and CPV-447 are presented in Table proteinsIn this study, a virus was isolated from infected minks from a commercial mink farm in China. The virus, MEV/LN-10, was identified as a mink enteritis virus, based upon its cytopathic effect in F81 cells, electron-microscopical structure, physicochemical, biological and serological properties and experimental infection studies in minks.In addition to high mutation rates and positive selection of mutations in the viral capsid gene, genetic recombination has also been determined to be an important factor in parvovirus evolution. ShackelWe analyzed the sites of key amino acids in the VP2 and NS1 proteins of the MEV/LN-10 strain and found that amino acids at positions 80, 93, 103, 323, 564 and 568 of VP2, which affect the host-range and antigenic properties of parvoviruses isolated from carnivores, are conThe mutations, at position 234 of VP2 protein and 540 of NS1 protein, are new and have not been reported previously in MEV. Further work is required to determine their significance in the infection of mink by MEV/LN-10. Four mutations in VP2 at residues 87, 101, 300, and 305 have been shown previously to alter host range, transferrin receptor binding, and antigenic structure in parvoviruses. For exaCPV-2 emerged as a new causative agent of severe enteritis in dogs in 1978, and it probably derived as a variant of FPLV or of a closely related virus infecting another carnivore. Subsequently, CPV-2 was replaced in nature by antigenic variants  which now coexist in dog populations worldwide,28. AfteIn a previous study, different evolutionary patterns of parvoviruses isolated from wild animals and non-domestic cats and dogs were described, highlighting the potential role of alternative hosts (e.g. raccoons) in providing indirect viral emergence pathways. Mink maThe authors declare that they have no competing interests.JK Wang wrote the manuscript and carried out the experiments with the help of S Yang who carried out virus isolation, L Yi contributed to the recombination analysis, SP Cheng carried out sequence analysis, YN Cheng carried out PCR and HL Xu participated in the sequence alignment. XJ Yan, H Zhao and H Wu revised the manuscript. All the authors have read and approved the final manuscript.


Daubenton bats may roost infrequently in human dwellings, so risk for human contact is low. We report the first seroprevalence study of the occurrence of specific antibodies to European bat lyssavirus type 2 (EBLV-2) in Daubenton's bats. Bats were captured from 19 sites across eastern and southern Scotland. Samples from 198 Daubenton's bats, 20 Natterer's bats, and 6 Pipistrelle's bats were tested for EBLV-2. Blood samples (N = 94) were subjected to a modified fluorescent antibody virus neutralization test to determine antibody titer. From 0.05% to 3.8%  of Daubenton's bats were seropositive. Antibodies to EBLV-2 were not detected in the 2 other species tested. Mouth swabs (N = 218) were obtained, and RNA was extracted for a reverse transcription–polymerase chain reaction (RT-PCR). The RT-PCR included pan lyssavirus-primers (N gene) and internal PCR control primers for ribosomal RNA. EBLV-2 RNA was not detected in any of the saliva samples tested, and live virus was not detected in virus isolation tests. Lyssavirus genus that with 5 other genera make up the family Rhabdoviridae within the order Mononegavirales. The Lyssavirus genus is differentiated into 7 genetically divergent lineages, Rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), EBLV-1 (genotype 5), EBLV-2 (genotype 6), and Australian bat lyssavirus (genotype 7). With 1 exception (Mokola virus), all remaining genotypes have been isolated from bats (Martes foina), and 5 cases in sheep (Ovis aries)  have been identified (>700 cases) in several European bat species (Myotis Daubentonii): a pregnant female in 1996 in Sussex . However, the latter is not indigenous to the British Isles, and Pond bat roosts have not been reported anywhere in the United Kingdom. Similar surveillance strategies have been used in other European countries  test and assessing oral swabs for the presence of lyssavirus RNA. The mFAVN test for EBLV-2 and polymerase chain reaction assays were developed as research techniques and used as surveillance tools after the death of a bat conservationist in 2002  was fitted for individual recognition. Mouth swabs  were taken from each bat with either a dry sterile swab or a combination of dry and wet sterile swabs. These were stored individually in 500-µL sterile transport buffer .Blood (up to 100 µL) was taken by puncture from the antebrachial or uropatagial veins  by using a 26-gauge needle and then collected by using 10- to 50-µL heparinized glass capillary tubes . A proprietary antibleeding product  was then applied to each puncture site. The capillary tubes were then emptied into a sterile screw-topped tube. To increase blood volume obtained, 50 bats were attached by elastic bands to a thin cork board that was placed on a heated surface (≈43°C) to ensure vasodilatation of the peripheral veins. All samples were refrigerated and stored at ≈4°C until testing (1–5 days later). Capturing, handling, ringing, and sampling of bats were done under guidelines approved by the Home Office (UK Project Licenses PPL 60/3122 and PPL 30/1948).From 25% to 35% of sampled bats were recaptured bats. Recapturing occurred within days of original sampling and also during a 1-year period from the date of initial sampling. This finding suggests that the sampling procedure was not harmful to bats. Ethical constraints in the United Kingdom restrict the resampling of wild bats within 3 months of capture. For this reason, additional sampling opportunities were limited.A fluorescent antibody test  is routinely used to measure levels of antibodies to rabies virus in sera from vaccinated animals by using rabies virus strain CVS. The mFAVN test used in this study was based on the routine test but used an EBLV-2 virus  ] and a pooled anti-EBLV-2 [inactivated RV628] serum from rabbits) and negative controls . A positive serum sample from an EBLV-2–infected bat was not available for full validation of this test.The mFAVN test is a quantitative procedure requiring a threshold to separate positive from negative results. To eliminate false-positive results, studies in Spain  needed for the test. Blood was pooled only across 1 species at any given site and with samples of a similar volume, such that plasma from an individual bat contributed equally to the pooled sample. As a result of pooling and being unable to determine the number of bats that were antibody positive or negative in a pooled sample, the 95% confidence intervals (CIs) were broader than if no pooling had taken place.-3 50% tissue culture infectious doses of rabies virus  only, while seropositive bats were tested by both the RTCIT and the mouse inoculation test (MIT) as previously described  was then used to generate the approximate 95% confidence limits by assuming 2 [ln L (p^) – ln L (p)] is approximately χ2 distributed. All calculations were programmed in R , and 8 Pipistrelle's bats (Pipistrellus species). Of these, blood from 224 bats was subjected to the mFAVN test. Fifty-five (24.5%) blood samples were tested individually; the rest were combined into pools containing 2–9 samples, with most containing 3 samples. The distribution of these samples across different sites is shown in the Blood was collected from 230 bats: 198 (85%) Daubenton's bats, 24 Natterer's bats  in which a successful positive or negative result was obtained. Positive samples were obtained in 4 pools  and 2 single samples, and were exclusively from Daubenton's bats caught at 2 sites . This fiHost rRNA was detected in 218 (65%) of the samples, indicating that saliva, cells, or both were present on the swab. In the remaining 35%, RNA was absent or below the limit of detection. No difference was detected in the ability to detect RNA when wet and dry swabs were compared. None of the results of the first-round or heminested PCRs with any of the samples were positive for lyssaviruses. These data suggest that none of the bats tested were actively excreting virus.Virus isolation tests were conducted using RTCIT  and MIT (antibody-positive bat swabs only). All RTCIT samples were negative and at day 41 after injection, clinical signs of infection had not developed in any mouse. These data indicate that live virus was not detectable in the oral swab samples.If the bats tested in this study (with the exception of those at site 1) were a truly random selection of Daubenton's bats across Scotland, the likely prevalence (95% CI ) of bats testing seropositive for EBLV-2 would be 0.05%–3.80%. The 95% CI for the prevalence of EBLV-2–seropositive bats at site 1 was 2.9%–16.3%.–Eptesicus fuscus)  and that active virus excretion is below the limit of detection used. If this prevalence of EBLV-2 was distributed evenly throughout Scotland, samples from large numbers of bats would be needed to refine this estimate. The effect of using the same detection threshold in the mFAVN test for both single and pooled samples may result in an underestimate of the number of antibody-positive bats.Antibodies to EBLV-2 in Daubenton's bats were found at only 2 of 19 sampled sites in Scotland. Whether the lower prevalence rates in Scotland will persist and are a function of either the bat or the virus being at the northerly edge of its range, or as suggested by a Spanish study , Rabies virus can elicit a measurable antibody response after exposure, but not all exposures are lethal; some lead to an abortive infection (In future studies, following up this research and successively resampling specific sites to establish disease profiles for ringed bats within this population will be important. To this end, blood samples of sufficient volume to permit individual tests must be obtained from seropositive bats over time at sites where positive cases have occurred  and at rThe available evidence suggests that the prevalence of EBLV-2 in Daubenton's bats in Scotland is low and may be sporadic (


The study aimed to determine costs to the state government of implementing different interventions for controlling rabies among the entire human and animal populations of Tamil Nadu. This built upon an earlier assessment of Tamil Nadu's efforts to control rabies. Anti-rabies vaccines were made available at all health facilities. Costs were estimated for five different combinations of animal and human interventions using an activity-based costing approach from the provider perspective. Disease and population data were sourced from the state surveillance data, human census and livestock census. Program costs were extrapolated from official documents. All capital costs were depreciated to estimate annualized costs. All costs were inflated to 2012 Rupees. Sensitivity analysis was conducted across all major cost centres to assess their relative impact on program costs. It was found that the annual costs of providing Anti-rabies vaccine alone and in combination with Immunoglobulins was $0.7 million (Rs 36 million) and $2.2 million (Rs 119 million), respectively. For animal sector interventions, the annualised costs of rolling out surgical sterilisation-immunization, injectable immunization and oral immunizations were estimated to be $ 44 million , $23 million  and $ 11 million (Rs 590 million), respectively. Dog bite incidence, health systems coverage and cost of rabies biologicals were found to be important drivers of costs for human interventions. For the animal sector interventions, the size of dog catching team, dog population and vaccine costs were found to be driving the costs. Rabies control in Tamil Nadu seems a costly proposition the way it is currently structured. Policy makers in Tamil Nadu and other similar settings should consider the long-term financial sustainability before embarking upon a state or nation-wide rabies control programme. Rabies is a fatal viral disease. It is transmitted mostly through dog bites in greater parts of Asia and Africa. It is primarily a disease of the poorer population groups with children being the most vulnerable. Control of rabies among humans therefore requires interventions in the animal as well as the human sectors. Animal sector interventions include vaccination accompanied with or without sterilization of dogs. Human interventions are limited to individual vaccination following dog bites. We estimated the costs to the government of rolling out animal as well as human sector interventions across an entire state having a human population of 72 million. We also estimated the major drivers influencing program costs and the implications to the government of adopting such a strategy over a long time. We found that the animal sector interventions were many times more costly than the most expensive human interventions. We also found that in the absence of dog population control measures, it will require substantial financial commitment on the part of the government to be able to invest in dog vaccination strategies. While rabies has been identified as a priority zoonoses that needs to be addressed globally Evidence from India and elsewhere demonstrates the efficacy of principle rabies intervention strategies. Indian researchers have studied the application of different post-exposure prophylaxis (PEP) regimens among humans However, as previously documented, rabies researchers have not been able to satisfy the information needs of policymakers Accordingly, we undertook a costing exercise building upon an earlier assessment Tamil Nadu is the southernmost state in India having a population of 72 million As described in Using program data from the earlier assessment in Tamil Nadu, we estimated the annual costs of scaling up those interventions across the state, including rural areas. An activity based costing approach was used. The interventions for human and animal populations were calculated separately and the costs for different components within these interventions were disaggregated. System-wide and environmental interventions, such as waste management and vaccine supply chain management systems were not included in the costing framework . Costs wBased upon the existing interventions in Tamil Nadu Based upon the feedback received from local program managers The annual number of outpatient visits for dog bites was calculated from the monthly dog bite visits reported by the state disease surveillance system over a twenty month period from January 2008 to August 2009. This was divided by the expected number of hospital visits for each dog bite case, to arrive at the annual number of dog bites in the state. While the national guidelines The then-prevalent model of ABC-AR was selected as one of the intervention strategies. Parenteral vaccination using teams of dog-catchers and oral vaccination were selected as hypothetical intervention scenarios to determine the extent to which costs could be reduced by less resource-intensive exercises.Using dog population density figures from the livestock census A base case scenario was constructed for each of the five different combinations of human and animal interventions using the existing or most likely estimates of key input parameters. The values of input data for our analysis were sourced from our review of program documents, published research literature and from our personal observations in the state. A sensitivity analysis was conducted by varying the values of principle input factors. More than 224,000 scenarios of animal and human interventions were tested. The values of input parameters for the base case and alternative scenarios have been described in Supplementary Files. These were refined based upon the feedback received from experts at two different national consultations of Indian rabies experts organized in 2011 Rabies control is a long term proposition, requiring sustained levels of high coverage of interventions in the animal populations We projected costs of four interventions—ABC-AR, injectable vaccination, oral vaccination, and a hypothetical intervention coupling injectable vaccination with injectable contraception for 20 years based on 2012 costs. For interventions involving contraception, a decrease in the dog population was estimated from a dog demographic model used earlier in India Since no other dog demographic models in the Indian context were available, we estimated the total number of stray dogs and its proportion that would be sterile for each year in Tamil Nadu assuming a similar setting and level of coverage. To project costs for future years, annualized capital costs (for 5-year depreciation) were assumed to be constant over 20 years, and recurrent costs were scaled to the projected dog population size in each year. Recurrent costs were calculated separately for the unsterile (requiring vaccination and sterilization) and sterile (requiring only vaccination) dog populations. Interventions which did not involve sterilization assumed a constant dog population. For the hypothetical injectable vaccination and contraception intervention, the additional cost of the injectable contraceptive was assumed to be negligible and the initial cost in 2012 was assumed to be the same as the cost of the injectable vaccine intervention alone in the base case scenario.The study is based upon one-time costs data collected from state programme managers. Therefore the analysis only considers those human cases that were reported to the public health surveillance system. This is likely to be an underestimate. Moreover, there is limited data on the completion of treatment; and it is possible that a small portion of patients might not complete their treatment, leading to a further underestimate of dog bite incidence rate. Data on categorization of dog bites, dog bite burden among animals and dog ecology is limited. In the absence of more data, the upper and lower bounds of the input parameters were taken from a range of sources, including expert opinion, summarised in Supplementary Files 1 & 2. In the absence of longitudinal data, we used dog demographic projections from an Indian study The annual costs of providing post exposure prophylaxis with antibodies for severe dog bites for Tamil Nadu was calculated to be $ 2.2 million . This waOn varying the key input parameters, we found that the costs of the human interventions ranged from $3–$82 million, while the costs for animal intervention ranged from $9–$98 million annually. In order to compare the relative effects of different cost components on individual set of interventions, a tornado chart was prepared  centred In the case of human interventions (Vaccine only and Vaccine+Antibodies), health seeking patterns, cost and wastage rates of vaccine and antisera and the burden of dog bites were found to be the major cost drivers causing the greatest fluctuations in the cost of the program. In relation to the other drivers, the use of intradermal versus intramuscular vaccine regimes did not greatly influence program costs. Antibody procurement comprised around 70% of total costs of the human sector interventions, followed by vaccine procurement costs and training & health promotion costs.In case of animal-sector interventions, the dog population and number of dog catchers required per team appear to be important drivers of ABC-AR programme costs. On the other hand, vaccine costs have greater role to play in influencing costs of vaccination-only programmes. Sex distribution of dogs does not affect total program costs in the long term, even for ABC-AR in which different surgical procedures are required for male and female dogs.Assuming an average sterilization rate of 62–87%, the dog population size in Tamil Nadu would be expected to decrease by 70% over a 20 year period from an estimated 2,022,055 dogs in 2012 to 615,408 in 2032 (Supplementary File 4). The proportion of sterilized dogs would stabilize at 80% such that the number of dogs needing ABC would decrease by 94% from 2,022,055 in 2012 to 123,082 dogs in 2032.Projected costs for ABC-AR, injectable vaccination, oral vaccination, and injectable vaccination-cum-contraception are shown in In keeping with assertions about rabies control being the responsibility of the local governments The identification of major drivers of programme costs can inform programme management by identifying areas to improve program efficiency and direct research efforts towards development of precise estimates where required. Basic epidemiological parameters such as incidence of dog bites, their categorization and dog population density were found to be among the major drivers of the program costs. These knowledge gaps require more attention from researchers and can be easily filled through focussed research studies. From the program perspective, the procurement and wastage rates of vaccine and antibodies were found to greatly influence total program costs. Strengthening of local procurement and supply chain management systems and negotiating long-term procurement rates are some of the options that could help offset these costs.Using base case scenarios, scaling up the existing animal interventions (ABC-AR) in Tamil Nadu would require 20 to 65 times the funds required for scaling up human post exposure prophylaxis alone. Moreover, a combination of human Post-exposure prophylaxis with ABC-AR would cost over 2.1% of the annual budgetary allocations for the departments of health, animal husbandry and municipal administration together in Tamil Nadu This is an important lesson for the proposed national rabies control programme in India which is currently structured around financing ABC-AR operations across selected cities Due to the challenges of achieving high vaccination coverage even among humans Antibodies were not made universally available for human vaccines because of the costs involved. Our calculations show that the costs of a combined (antibody plus vaccine) rabies programme would be three times the costs of vaccine only intervention and is likely to cost an additional expenditure of $ 1.5 million (Rs. 82.5 million) annually.Given the costs of different vaccine formulations in Tamil Nadu, choosing intradermal over intramuscular vaccine regimen is likely to result in annual savings of $ 13,000  only. This relatively small amount should not be a deterrent to state public health programme managers in choosing a vaccine regimen that is more appropriate to the clinical setting and qualifications of their staff Rabies control efforts in Tamil Nadu seem a costly proposition as they are currently structured in the state. This would necessarily require high levels of technical, political and financial commitments before the government chooses to embark upon a long-term rabies control strategy. Given recent recognition of the need for a national rabies control programme in India by the National Centre for Disease Control Supporting Information S1Input factors used for the cost analysis of Human Interventions.(DOCX)Click here for additional data file.Supporting Information S2Input factors used for the cost analysis of Animal Interventions.(DOCX)Click here for additional data file.Supporting Information S3Breakup of animal intervention costs.(DOCX)Click here for additional data file.Supporting Information S4Program-adjusted rates of ABC-AR.(DOCX)Click here for additional data file.Supporting Information S5Dog demographic model.(DOCX)Click here for additional data file.Supporting Information S6Calculations.(XLSX)Click here for additional data file.