Neurologic manifestations of Zika infection must be adequately recognized and treated; our study methods can be used for monitoring and warning systems. Evidence is increasing that Zika virus can cause extensive damage to the central nervous system, affecting both fetuses and adults. We sought to identify traces of possible clinical manifestations of nervous system diseases among the registers of hospital admissions recorded in the Brazilian Unified Health System. Time series of several diagnoses from the International Classification of Diseases, 10th Revision, were analyzed by using control diagrams, during January 2008–February 2016. Beginning in mid-2014, we observed an unprecedented and significant rise in the hospitalization rate for congenital malformations of the nervous system, Guillain-Barré syndrome, encephalitis, myelitis, and encephalomyelitis. These conditions are compatible with viral infection and inflammation-associated manifestations and may have been due to the entrance of Zika virus into Brazil. These findings show the necessity of adequately diagnosing and treating suspected cases of Zika virus infection and also that health surveillance systems can be improved by using routine data. Aedes, such as dengue, yellow fever, and chikungunya viruses.The recent spread of Zika virus across the globe has worried citizens and public health authorities. In late February 2016, the World Health Organization (WHO) declared an international state of emergency because of the advance of microcephaly associated with the Zika virus , leading to certain congenital malformations  is primarily financial and administrative in nature, but it can be used as a data source for epidemiologic studies and surveillance because of advantageous characteristics, such as national coverage, relative consistency and standardization in terms of data generation, and rapid production and dissemination of data. These features facilitate its incorporation into epidemiologic alert systems, especially those for infectious diseases.An analysis of hospital admissions data can provide a baseline that enables identification of trends in the number of admissions for neuropathies that affect the nervous system, as well as inflection points that depart abruptly from these trends. Long-term analyses of diseases potentially associated with Zika virus continue to be nonexistent in Brazil and have become urgently needed to bring into focus the scope of the problem, as well as delineate monitoring strategies for the epidemic in Brazil and other affected countries.The main objective of this study was to analyze temporal patterns in hospitalizations in separate Brazilian regions during January 2008–February 2016, for neuropathies, which could have been associated with Zika virus infection in separate Brazilian regions. This period includes the time before the epidemic and the onset of the Zika epidemic in Brazil.This is a descriptive study of a time series from a population dataset. We sought to evaluate historical trends in neurologic conditions potentially related to infection with Zika virus, based on the principal diagnosis assigned at discharge as the main reason for hospitalization. Data were made available by the Informatics Department of the National Health System through the Hospital Information System during January 1, 2008–February 29, 2016, according to each patient’s state of residence.We selected the health problems analyzed in this study on the basis of monitoring protocols put in place to track reports of microcephaly related to infection with Zika virus . With respect to congenital malformations of the nervous system in children <1 year of age, we calculated the rate of hospitalizations using the number of live births per month as the denominator. In the case of abortion, we obtained the rate of hospitalizations using the number of live births per month as the denominator with a lag of 7 months, which reduced the effect of seasonality so common in data for births as well as for abortions.The total number of hospitalizations can vary greatly by state due to variations in staffing at the local level and disparities in access to services for utilizers of the public health system  as upper limits for detection of peaks in the time series. These data were plotted as control charts, which allowed for evaluation of long-term trends, seasonality, and anomalies in the series by visual inspection. We tested the difference between observed and expected number of hospitalization—assuming the historical baseline as a control parameter—by calculating the rate ratio (IRR) and CI using Poisson regression.The rate of hospitalizations for congenital malformations of the nervous system (ICD-10 Q00-Q07) presented a stable mean value of 40/100,000 live births in the Northeast region until September 2015. As of November 2015, however, we observed an increase in this rate, which, in the last months of the series, reached a mean value of 170 hospitalizations for congenital malformations of the nervous system per 100,000 live births, an increase ≈4 times higher than historical rates . In February 2016, all regions exhibited rates of ≈100 hospitalizations for congenital malformations of the nervous system per 100,000 live births, a 2-fold increase over the national historical baseline. From November 2015 through February 2016, a total of 1,027 hospitalizations for congenital malformations of the nervous system were recorded nationwide; 448 of these occurred in the Northeast region. This region is responsible for ≈830,000 (28%) of the overall annual live births in Brazil.In the Northeast region, the hospitalization rate for GBS (ICD-10 G610) was 0.05/100,000 residents until May 2015, when an outbreak occurred, which peaked in July 2015. From June 2015 through February 2016, the hospitalization rate was 0.11/100,000 residents, an increase by a factor of 2.7 (95% CI 2.5–3.0). During July through October 2015, 377 GBS hospitalizations were recorded in the Northeast region, an excess of ≈240 hospitalizations in the region.The hospitalization rate for CNS inflammatory diseases, represented by encephalitis, myelitis, and encephalomyelitis (ICD-10 G040-G049), increased in the Northeast region, principally as of September 2014, from a baseline of 0.05 hospitalizations/100 residents to 0.11 hospitalizations/100,000 residents . In the rest of the country, a stable base line of 0.05 hospitalizations/100,000 residents was observed. From September 2014 through February 2016, hospitalizations for encephalitis, myelitis, and encephalomyelitis in the Northeast region reached 1,115, an excess of ≈570 hospitalizations.With respect to GBS , panel BA historical series of the rates of hospitalization for inflammatory diseases of the CNS, represented by encephalitis, myelitis, and encephalomyelitis, shows a pattern change in recent years , panel CWe did not observe changes for abortions and related problems in the times series, including for spontaneous abortions (results not shown). We also did not observe changes in the total number of hospitalizations or in hospitalizations for nervous system problems  in the regions during the study period. This pattern may demonstrate the stability of the health system in terms of specialty beds, especially for neurology.In this study, we analyzed time trends for hospitalizations for some neuropathies. We found increases in the number of hospitalizations for congenital malformations of the nervous system, GBS, and some inflammatory diseases of the CNS. These complications began to cause more hospitalizations, with strong fluctuations over the course of the study period, beginning even before the first warning in November 2015 about the possible effects of infection with Zika virus on microcephaly and other malformations. These shifts from the previous baseline were not, therefore, influenced by new procedures and norms that have been incorporated into the health system over the last months. As such, the complications that we present here can be thought of as early signals of neurologic complications that were unperceived through health surveillance systems, but that may be related to the entrance and circulation of the Zika virus in Brazil, especially in the Northeast region.Our results show that the rates of hospitalization in the Northeast region for some neuropathies were subject to abrupt changes beginning in mid-2014, when it is believed that Zika virus was introduced to the country , when in general the incidence of vector-transmitted diseases, such as dengue, is lower because of milder temperatures . However, because the first alerts about microcephaly related to Zika virus infections were issued in November 2015, and only in February 2016 did WHO declare a public health emergency, the previous months’ records may have not been affected by health surveillance guidelines, with the exception of microcephaly.A second limitation concerns the fact that the health information system for hospitalizations registers events and not individual patients. As such, rehospitalizations can be contained within that figure, leading to overestimation of hospitalization rates. These 2 limitations likely do not substantially affect the results of this study because they would have had to occur uniformly across the nation in the period studied.This study can contribute to a greater understanding of the Zika epidemic in Brazil and its additional effect on certain neuropathies. The data sources and methods we used in this study can be used for monitoring and warning systems in cases of emergency. Implementation would require obtaining and statistically analyzing time series data, to establish a baseline of adverse events and identify peaks that could represent outbreaks of disease.To monitor the effects of Zika virus, public health researchers must consider its various clinical manifestations that may be registered in different ICD codes, even in dispersed chapters, such as viral encephalitis (ICD-10 A83) and provisional code (ICD-10 U06), as recommended by WHO. The hospital routine may account for the diversity of codes used to describe complications derived from Zika virus. The admission of a patient to the hospital is first justified by a preliminary ICD code. Throughout the period of hospitalization, new examinations and procedures are carried out, which can alter this classification. At the end of hospitalization period, a code for the principal diagnosis is assigned to the outpatient, based on procedures performed and their costs, which is not necessarily related to the disease etiology. A wide range of ICD codes should be taken into account in this situation, with anticipation of all possible reasons for hospital admission potentially related to Zika complications.New detection tools for outbreaks and means of communicating warnings should be pursued to identify real trends and, at the same time, minimize false alarms and panic that could be provoked in populations potentially affected by the Zika epidemic. The history of neuropathies caused by viruses is about to change, not only because of the effects that Zika virus has been found to have on the CNS, but also because of the effort of gathering evidence that will help construct this history.


IAS) eradications from small islands (<10 km2), but few from more extensive areas. We review 15 large‐scale removals (mean area 2627 km2) from Northern Europe since 1900, including edible dormouse, muskrat, coypu, Himalayan porcupine, Pallas' and grey squirrels and American mink, each primarily based on daily checking of static traps. Objectives included true eradication or complete removal to a buffer zone, as distinct from other programmes that involved local control to limit damage or spread. Twelve eradication/removal programmes (80%) were successful. Cost increased with and was best predicted by area, while the cost per unit area decreased; the number of individual animals removed did not add significantly to the model. Doubling the area controlled reduced cost per unit area by 10%, but there was no evidence that cost effectiveness had increased through time. Compared with small islands, larger‐scale programmes followed similar patterns of effort in relation to area. However, they brought challenges when defining boundaries and consequent uncertainties around costs, the definition of their objectives, confirmation of success and different considerations for managing recolonisation. Novel technologies or increased use of volunteers may reduce costs. Rapid response to new incursions is recommended as best practice rather than large‐scale control to reduce the environmental, financial and welfare costs. © 2016 Crown copyright. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Numerous examples exist of successful mammalian invasive alien species ( A review of the effects of IAS from different taxonomic groups on ecosystem servicesThe most effective way to manage the impacts caused by biological invasions is the prevention of new unwanted introductions, but once this has failed, early eradication is the best alternative, considering the costs and undesired effects related to permanent control or to a ‘do nothing’ policy.Most documented eradications relate to islands, with 89% of reported European eradicationsEradication has been defined as the complete and permanent removal of all wild populations of an alien plant or animal species from a defined area by means of a time‐limited campaign.New European legislationGreat Britain, Ireland and Belgium have a long history of controlling large‐scale mammalian IAS, starting in 1900. These programmes, conducted at a regional or national level, provide insights into the costs and effectiveness of control in larger land masses. They have also demonstrated a variety of objectives, reflecting different approaches to the challenges of control over large areas. Although described in the literature, the costs and effectiveness of these large programmes have not been presented in a consistent format. In this paper we present data on the effort and associated costs encountered, and contrast these with the published literature on smaller areas, typically islands. We discuss the challenges associated with widespread control over larger land areas and the range of objectives applied, together with novel approaches to reducing costs and improving success.22) land areas, typically islands, as these have been documented elsewhere.Glis glis, L.), muskrat , coypu , Himalayan porcupine , American mink , grey squirrel  and Pallas' squirrel . In addition, the programme to remove the resident breeding ruddy duck  population from the United KingdomWe searched the scientific literature and consulted with experts to identify programmes that have attempted the eradication or complete removal of mammalian IAS from large contiguous land masses in Northern Europe. We did not consider projects based on small , grey squirrels, mink and coypu, based on government support for local control groups or provision of bounties.Muntiacus reevesi, Ogilby), muskrat and racoon  are subject to ongoing control, but without eradication as the aim.In Great Britain there were also a number of historic schemes for the local control of rabbits  were considered successful. Data on worldwide island eradications were used for comparative purposes. Details are available of the costs of 41 eradications of a range of mammal species on islands of varying sizes,The details of the 15 large‐scale eradication/removal programmes are presented in Table n = 11, F = 73.8, P < 0.001, r2 = 0.879)  nor the addition of the year of completion (P = 0.833) had any significant effect on the relationship.The manpower estimates (log) for the 11 successful large‐scale programmes for which data on effort were available were entered into a model including area (log), number of animals removed (log) and year completed. This identified a significant relationship between the area over which removal took place and the reported manpower to achieve this . The eradication programmes from larger land masses were significantly larger, but there was no evidence that the costs of island or large‐scale programmes differed in their relationship with area (P = 0.201).Combining the estimated costs of these large eradications (using the median estimate of $US 100 k per man‐year of effort) with the reported costs of small island eradications from the literaturen = 51, F = 41.9, P < 0.001, r2 = 0.456). There was no evidence that the cost per unit area of island or larger‐scale programmes differed in their relationship with area (P = 0.201)  Fig. .42). The examples described here comprise a significant proportion of such large control programmes, for Europe at least, and these have been orders of magnitude larger than those on islands worldwide. Nevertheless, the effort required to achieve eradication in these large programmes appeared to follow a similar form to that observed on islands.The total number of large‐scale mammalian IAS programmes is small, and certainly considerably fewer than have taken place on islands  persistence, although this requires continued resource input.On Anglesey, grey squirrel control eradicated this species from the island itself, but ongoing control in a 2 km wide and 5 km long adjacent coastal mainland area was necessary to prevent individuals from crossing the narrow sea‐strait via road or rail bridges and recolonising Anglesey.2 to reduce grey squirrel abundance to low levels and ideally to zero within a central 17 km2 forest area, using approximately 0.5 man‐years of effort per year. However, the number of grey squirrels in the core area continued to increase through time, and thus the level of control was not sufficient to prevent grey squirrel occupation.The resource challenges posed by grey squirrel control are reflected in a useful example of an experimental programme that failed to achieve landscape clearance but quantified control effort and returns. This 3 year control programme was conducted in Thetford forest, East Anglia, a managed coniferous plantation that contained a nationally important remnant red squirrel population vulnerable to competitive replacement by the congener.Herpestes javanicus, St‐Hiliare) control on OkinawaSus scrofa, L.) from fenced areas in US national parks.There are additional examples of the successful use of such intermediate strategies, removing mammalian IAS to a barrier or maintaining a buffer zone to prevent recolonisation. These include the use of fenced ‘mainland islands’ in New Zealand,Ongoing control within an area to reduce abundance, associated damage and the risk of spread is widely undertaken by local interests, for example predator removal by gamekeepers, deer management and local pest control. It does not specifically include complete local removal or eradication as an objective, and effort is often determined by local perceptions of damage or financial incentives for the activity. In Great Britain, government funds were used to support such local activities between 1950 and 1970 in relation to coypu, mink, rabbit and grey squirrel control, providing funds for local control groups or bounties.Castor canadensis, Kohl) population from FranceErinaceus europaeus L.) is ongoing,Myiopsitta monachus, Boddaert) from southern England.While coordinated databases of island eradications are maintained,In spite of these large‐scale programmes spanning 11 decades, there was no suggestion that efficiency of programmes based on the employment of trappers improved over that period, and the basic approach of daily checking of static live‐capture or lethal traps remained unchanged. The majority of the cost of such programmes comprises the manpower costs of the trappers. More recently, the Scottish Mink Initiative and regional grey squirrel control have relied on public volunteers to undertake much of the trapping. This approach greatly reduces the manpower costs compared with dedicated teams of trappers and has been successful in mammalian IAS removal over large areas, but current examples require continued inputs at their borders and to remove dispersing animals. The use of this voluntary approach to achieve permanent eradication has yet to be demonstrated but has clear potential for some species and habitats. New technologies also offer the prospect of more labour‐efficient approaches. Examples include self‐reporting or self‐resetting traps,Lastly, these eradications have proven to be considerably geographically larger than is the case for islands, with greatly increased total costs as a consequence, although mitigated to some extent by the economies of scale described here. Although accepted best practice is for there to be a rapid response to remove new invasive species,


In January 2016, a combined outbreak of highly pathogenic (HP) avian influenza virus (AIV) and low pathogenicity (LP) AIV occurred in commercial turkeys in the state of Indiana, United States. Genetically, the viruses were highly similar, belonged to the North American wild bird lineage, and had not been previously detected in poultry. In order to understand the pathobiology of the H7N8 LPAIV and HPAIV, infectivity, transmission and pathogenicity studies were conducted in chickens, turkeys, and mallards. Among the three species the lowest mean infectious dose for both the LP and HP phenotype was for turkeys, and also disease from the LPAIV was only observed with turkeys. Furthermore, although the HPAIV was lethal for both chickens and turkeys, clinical signs caused by the HPAIV isolate differed between the two species; neurological signs were only observed in turkeys. Mallards could be infected with and transmit both viruses to contacts, but neither caused clinical disease. Interestingly, with all three species, the mean infectious dose of the HP isolate was at least ten times lower than that of the LP isolate. This study corroborates the high susceptibility of turkeys to AIV as well as a pathobiology that is different from chickens. Further, this study demonstrates that mallards can be asymptomatically infected with HP and LP AIV from gallinaceous poultry and may not just be involved in transmitting AIV to them. Wild aquatic birds are the natural reservoirs of avian influenza virus (AIV) and usually carry the low pathogenic (LP) phenotype . PeriodiOn January 16, 2016, an H7N8 HPAIV infection was confirmed in a turkey flock on a single farm in the state of Indiana, United States (US), and LPAIV of the same subtype was subsequently detected in nine nearby turkey flocks . InitialGallus gallus domesticus], turkeys [Meleagris gallopova] and mallards [Anas platyrhynchos]) ) . In thisEgg passage 1 stocks of A/turkey/Indiana/16-001571-6/2016 (H7N8) LPAIV (GenBank accession numbers KY684308-KY684315) and A/turkey/Indiana/16-001403-1/2016 (H7N8) HPAIV (GenBank accession numbers KU558903-KU558910 [rd Edition. Humane endpoints were used and euthanasia was the primary method to minimize suffering and distress. Euthanasia is applied to birds that are moribund. Birds are considered moribund if they meet one or more of the following criteria: 1) impaired ambulation which prevents animals from reaching food or water; 2) lack of physical or mental alertness; 3) difficult, labored breathing; and 4) inability to remain upright. Turkeys and chickens were euthanized by cervical dislocation. Mallards were euthanized by intravenous administration of sodium-pentobarbital 100mg/Kg body weight. Animals were monitored twice daily when disease was apparent and once daily when the birds were healthy. No unexpected deaths or clinical signs were observed.Day-old commercial broad-breasted white turkeys and mallards were obtained from commercial producers and reared in animal biosafety level (ABSL) 2 enhanced facilities at the US National Poultry Research Center, USDA-ARS (USNPRC). Specific pathogen free (SPF) white-leghorn chickens were obtained from USNPRC in-house flocks. At 2 weeks of age  or 3 weeks of age, (chickens and turkeys) birds were transferred to ABSL-3 enhanced facilities for challenge. Ten chickens, turkeys and mallards were bled immediately prior to challenge to confirm the absence of AIV antibody with a commercial ELISA . Each experimental group was housed in self-contained isolation units ventilated under negative pressure with inlet and exhaust HEPA-filtered air. This study and associated procedures were reviewed and approved by the USNPRC Institutional Animal Care and Use committee. The study was conducted in accordance with the recommendations of the Federation of Animal Science Societies Guide for the Care and Use of Agricultural Animals in Research and Teaching, 36 50% egg infectious doses (EID50) (high dose), 104 EID50 (medium dose), and 102 EID50 (low dose) in 0.1ml and was administered by the intra-choanal route. The actual titers of the inocula were confirmed by titration in ECE and were within 0.5 log10 of the target titer for all groups.”Sham exposed birds were inoculated with 0.1 ml of sterile allantoic fluid diluted 1:300 in BHI media. To evaluate transmission by contact exposure, non-inoculated hatch-mates (contacts) were added to each dose group 24 hours (hr) post inoculation (PI).Chicken and turkey studies were conducted identically and simultaneously (i.e. the same inocula preparations were used for both species). Mallard studies were conducted with inoculum prepared at a different time and with a few minor modifications because of the expected differences in AIV pathobiology in the mallards, as noted below. Birds were divided into groups as shown in Oropharyngeal (OP) and cloacal (CL) swabs were collected from directly inoculated chickens and turkeys in the high dose group at 12hr, 24hr, 36hr, 48hr, 3 days (d), 4d, 7d and 10d PI. Oropharyngeal swabs included the buccal cavity and choanal cleft. Swabs (OP and CL) were collected from directly inoculated chickens and turkeys in the medium and low dose groups at 24hr, 48hr, 3d and 4d PI. Oropharyngeal and CL swabs were collected from contact chickens and turkeys at 24hr, 48hr, 3d and 4d after placement with the inoculated birds. Oropharyngeal and (CL) swabs were collected from mallards at 2, 4, 7, and 10d PI. Swabs were placed in 2ml of BHI broth with penicillin , gentamicin  and amphotericin B . The swab material was stored at -80C until it was processed.At 48hr PI two inoculated chickens and turkeys in the high dose group were euthanized and necropsied to examine for gross lesions and collect tissues for microscopic evaluation and visualization of viral antigen by influenza specific immunohistochemical (IHC) staining. Two sham exposed hatch-mates were also euthanized and necropsied as control birds. At 3d PI, three inoculated and 2 sham-inoculated mallards were euthanized for necropsy. A full set of tissues was collected from each bird and fixed in 10% neutral buffered formalin solution, paraffin-embedded, sectioned, and stained with hematoxylin-and-eosin. Duplicate sections were stained by IHC methods to visualize the distribution of influenza virus antigen in individual tissues [Body temperatures and body weights were taken from inoculated mallards in the high dose groups and from sham-inoculated mallards at 2 and 4d PI.Sera were collected from all surviving birds at 14d PI to evaluate infection status by antibody levels using hemagglutination inhibition (HI) assay. Hemagglutination assay was performed using standard methods and homologous antigen .The virus infectious dose was calculated by the Reed-Muench method , using t1.6 EID50/ml (chicken and turkey samples), and 101.9 EID50/ml . The threshold of detection in tissue samples was 101.5 EID50/g.Total RNA from swabs and tissues was tested by quantitative real-time RT-PCR (qRRT-PCR) targeting the influenza M gene as previously described . Virus t10 below the qRRT-PCR test limit of detection. Proportions of birds shedding were tested with Fisher’s exact test (SigmaPlot 13.0). A p value of ≤0.05 was considered significant.Shed titers by time point for the same swab type (OP or CL) were analyzed between the HPAIV and LPAIV and among species with the Mann-Whitney Rank Sum test . If virus was not detected in a sample, it was given the value of 0.1 log50) was 104.0 EID50 . Virus was detected at all swab sample collection times from the high dose group  and from50 was 103.6 EID50 ; and 80% and 100% were infected in the medium and high dose groups, respectively, therefore the BID.5 EID50 . One con.5 EID50 . Viral aSimilar to the LP isolate, mallards shed virus by both the OP and CL route with prolonged shedding by the CL route .Oral and CL shed titers between LPAIV and HPAIV infected birds were compared among all 3 species in the high dose groups. In chickens, HPAIV titers were significantly higher than LPAIV orally from 12 to 48hrs PI and cloacally from 24 to 48hrs PI . In turkWith both chickens and turkeys the proportion of birds shedding virus cloacally was significantly higher in the HPAIV exposed birds at 24, 36, 48 and 72 hr PI and statistically equal at 12hr PI (too few HPAIV exposed birds survived for comparison at later time points). With oral shed, the only time when the proportion of birds shedding virus was significantly different between HPAIV and LPAIV was at 48hr PI when more HPAIV inoculated chickens were shedding virus. In mallards there were no differences between the AIV phenotypes in the proportion shedding virus.The shed titers within a pathotype were compared among chickens, turkeys and mallards for both LPAIV and HPAIV. With LPAIV, chickens shed significantly more virus orally than turkeys at 12hr PI, but turkeys shed significantly higher titers at every other time point through 7d PI . TurkeysThe proportion of birds shedding had a similar pattern to shed titers. With LPAIV, significantly more chickens were shedding at 12hr PI, and significantly more turkeys were shedding virus at 48hr, 72hr, 96hr and 7d PI. More mallards than chickens were shedding LPAIV orally  and cloacally . By the CL route the only difference was at 7d PI when significantly more turkeys were shedding than chickens. With HPAIV, significantly more chickens were shedding at 12hr by the oral route and at 24hr PI by the CL route.At all other times the highest proportion shedding virus were either the turkeys or mallards. The overall trend was that mallards and turkeys were similar with more birds shedding virus than chickens.10 lower than the observed with the HPAI virus. In mallards, LPAIV was only detected in the brain and muscle of one mallard of three examined. Moderate to high HPAIV titers were detected in all tissues of all three mallards examined  [Clinical signs also varied among the three species. Neither chickens nor mallards exhibited clinical signs when infected with LPAIV, but turkeys presented with respiratory disease. Although the severity of disease was mild-to-moderate among the turkeys in our study, infection of turkeys in the field could be exacerbated by secondary infection or environmental stress –16. Prever bird) . A thirder bird) .6 EID50/g are typically required to be able to visualize the virus by this IHC method in tissues).Although the outcome of AIV exposure is reliant upon the specific isolate-species combination, in general and as corroborated in this study, turkeys appear to be more susceptible to AIV infection than chickens or mallards. In our study, with some exceptions (e.g. the lung and comb) broader tissue tropism and higher viral titers where observed in the turkeys when compared to chickens and mallards, as seen by both IHC and qRRT-PCR. Interestingly, LPAIV was detected by qRRT-PCR in several internal organs in infected turkeys as well. The specific cells in which the virus was replicating within these tissues could not be identified by IHC because of the insensitivity of this assay (titers of ≥10Anas platyrhynchos domesticus), with the exception of some strains of the A/goose/Guangdong/1/1996 lineage of H5 HPAIVs [The HPAIV isolate did not cause clinical disease in mallards, which is expected because most HPAIV strains do not cause disease in mallards or their domestic counterpart, Pekin ducks  [50 was at least 10 times lower for HPAIV than LPAIV for all three species. Increased shed titers and/or the proportion of birds shedding virus, which are consequences of increased efficiency of virus replication, could contribute to better transmission in the field. Transmission was not efficient among chickens in our study, which is likely an artifact of our housing (isolators with high rates of airflow and grate floors) and has been seen previously [The infectivity and intraspecies transmission of the viruses varied among the three species. Chickens were the most resistant to infection with either the LP or the HPAIV, and turkeys were the most susceptible, based on BIDisolates , 30–34. 2 LPAIV) . Here, teviously .In contrast to the H5N2 HPAIVs that caused a much more widespread outbreak in turkeys and layer chickens in the Midwest US in 2015, when over 200 farms were infected , the MDTInterestingly, in mallards, the HP isolate also had a lower mean infectious dose than the LP isolate. Recent reports demonstrate that mallards are susceptible to HPAIV infection ; howeverS1 FigA) Oro-pharyngeal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 17); B) Cloacal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 17). Bars represent mean and standard deviation; a dotted line represents the approximate limit of detection; samples where virus was not detected are shown at the limit of detection; LP = low pathogenic (shown in black), HP = highly pathogenic (shown in red). The 10day time point is not shown because insufficient chickens in the HP group were alive for statistical analysis. Brackets with an asterisk denote statistical significance at a p value of ≤ 0.05 between the bracketed groups.(PDF)Click here for additional data file.S2 FigA) Oro-pharyngeal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 17); B) Cloacal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 17). Bars represent mean and standard deviation; a dotted line represents the approximate limit of detection; samples where virus was not detected are shown at the limit of detection; LP = low pathogenic (shown in black), HP = highly pathogenic (shown in red). The 96hr, 7day and 10day time points are not shown because insufficient turkeys in the HP group were alive for statistical analysis. Brackets with an asterisk denote statistical significance at a p value of ≤ 0.05 between the bracketed groups.(PDF)Click here for additional data file.S3 FigA) Oro-pharyngeal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 8); B) Cloacal swabs from low pathogenic and highly pathogenic avian influenza virus exposed birds (n = 8). Bars represent mean and standard deviation; a dotted line represents the approximate limit of detection; samples where virus was not detected are shown at the limit of detection; LP = low pathogenic (shown in black), HP = highly pathogenic (shown in red). Brackets with an asterisk denote statistical significance at a p value of ≤ 0.05 between the bracketed groups.(PDF)Click here for additional data file.S4 FigA) Oro-pharyngeal swabs; B) Cloacal swabs. Bars represent mean and standard deviation; a dotted line represents the approximate limit of detection; samples where virus was not detected are shown at the limit of detection. Brackets with an asterisk denote statistical significance at a p value of ≤ 0.05 between the bracketed groups.(PDF)Click here for additional data file.S5 FigA) Oro-pharyngeal swabs; B) Cloacal swabs. Bars represent mean and standard deviation; a dotted line represents the approximate limit of detection; samples where virus was not detected are shown at the limit of detection. Columns with no data mean that there were no birds alive in the group at that time point. Brackets with an asterisk denote statistical significance at a p value of ≤ 0.05 between the bracketed groups.(PDF)Click here for additional data file.


Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host–virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 (ne219) strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 (ne219) mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 (ne219) mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear.  Caenorhabditis elegans (C. elegans) nematodes in rotting fruit in Orsay, France. It is the only identified virus capable of naturally infecting C. elegans  in the RNA2 segment  and Pseudomonas mendocina (P. mendocina), were found to confer resistance to the nematode pathogen Pseudomonas aeruginosa (P. aeruginosa). The protection offered by P. mendocina was p38-dependent (Bacillus nematocida (B. nematocida) B16 infection  or replication of flock house virus (FHV) in whole animals  recruits Dicer-1 and the dsRNA-binding protein RDE-4 (RNA interference-deficient 4) to recognize the viral dsRNA replication intermediate. Dicer-1, with the aid of its partner RDE-4, cleaves the viral genome into 23 nt viral siRNA duplexes with a 2 nt 3′ overhang  mutants.In addition to immune regulation, whether the intestinal microflora is involved in synergistic resistance to virus infection is unclear. The genes involved in the interaction with the intestinal microflora following virus infection virus remains unknown. In this study, we investigated the change in the intestinal microbiota community and the expression of related genes during the antiviral response of C. elegans, N2 strain and the RNAi deficient mutant WM27 rde-1 (ne219) was used in this study. All worm strains were maintained on nematode growth medium (NGM) plates at 20°C and seeded with the Escherichia coli (E. coli) bacteria strain OP50 unless otherwise indicated (C. elegans N2 and rde-1 (ne219) strain was conducted by transferring one square centimeter of agar with mixed stage animals onto a fresh OP50 seeded NGM plate.The Bristol isolate of ndicated . Serial Eggs were obtained by incubating freshly starved larvae on new 90 mm diameter seeded NGM plates with basic hypochlorite solution as previously described . SynchroC. elegans at room temperature and the virus filtrate was prepared following a protocol described previously (g for 5 min (4°C), the virus-containing supernatant was then filtered through a 0.22-μm filter unit . For all strains, 10 young adults were inoculated on 55 mm culture plates. At the same time, 40 μl of infectious filtrate was pipetted onto the bacterial lawn. The cultures were incubated at 20°C for 4 days. Infections were performed in four biological replicates.Orsay virus was maintained using the JU1580 isolate of eviously . To prepFour days after infection, all worms were collected in M9 and then divided into two parallels. The worms were treated based on the method described in the literatures . The wor® Blood&Tissue Kit  according to the manufacturer’s instructions. The genomic DNA was evaluated with a NanoDrop spectrophotometer , using an A260/A280 ratio between 1.8 and 2.0 as a criterion for quality control and agarose gel electrophoresis, respectively. DNA samples were stored at -20°C until use. Total RNA was prepared using Invitrogen TRIzol , by following the manufacturer’s instructions.DNA was extracted using the DNeasyTM Deep Well Real-Time PCR Detection System . Each sample was normalized to ama-1, and then viral RNA1 (primers GW194: 59 ACC TCA CAA CTG CCA TCT ACA and GW195: 59 GAC GCT TCC AAG ATT GGT ATT GGT) levels were compared to those uninfected animals as negative controls.cDNA was generated from 1 μg total RNA applying random primers using Superscript III , and cDNA was diluted to 1:100 for qRT-PCR analysis. qRT-PCR was performed using SYBR Green realtime PCR master Mix . The amplification was performed on a CFX96 Touchrde-1 (ne219) mutant animals were detected by single molecular fluorescent in situ hybridization (smFISH), which was performed as previously described  labeled with Quasars® 670 Dye for the Orsay virus RNA1 molecule in this experiment. The oligonucleotide sequences were reported by Accumulation of Orsay virus and the intestinal cell infection phenotype in N2 and escribed . We usedDNA libraries were constructed according to the standard protocol provided by Illumina. Quality control of libraries were performed before sequencing. Libraries were sequenced using the Illumina Miseq instrument  with 2 × 300 base pairs (bp) paired-end (PE) sequencing.mRNA was concentrated from total RNA and cDNA libraries were constructed using mRNA. After validation and the quality assessment of each library on gel electrophoresis and bioanalyzer , the libraries were sequenced on the Illumina Hiseq platform  with 2 × 150bp paired-end (PE) sequencing.The raw data generated by sequencing were analyzed using the pipeline tools in QIIME . Sequencde novo OTU-picking strategy. Taxonomies were assigned according to the Greengenes database.Data were de-noised by removing reads with average quality values <30. After removing both the barcodes and primer sequences, the filter-passed reads were clustered into Operational Taxonomic Units (OTUs) at a 97% similarity cutoff using the Following construction of the OTU table and phylogenetic tree, microbial richness ), diversity (Shannon Index and Simpson Index), and evenness  estimators were calculated. Besides, microbial structure was evaluated by analyzing weighted UniFrac distance, unweighted UniFrac distance, and Bray–Curtis distance metrics . The relQ ≤ 5) were removed. After which, the resulting filtered reads were mapped and annotated. Gene expression was quantified as Reads Per Kilo bases per Million reads (RPKM) (Raw sequencing reads were trimmed using the following criteria: (1) reads containing adapters were removed; (2) reads containing greater than 10% N bases were removed; and (3) reads containing greater than 50% low-quality reads (s (RPKM) . DESeq (s (RPKM)  was usedt-tests with 1000 permutations. (2) Differences in relative abundances of individual OTUs and microbial taxa  were analyzed using LEfSe  score > 2.0].The data were further analyzed with the following statistical methods. (1) Differences in alpha- and beta-diversity were evaluated using the non-parametric two-sample ng LEfSe  with defC. elegans upon Orsay virus infection, we analyzed 16S ribosomal DNA library data of infected and non-infected worms. The laboratory reference strain N2 and an rde-1 (ne219) mutant deficient in the primary Argonaute protein RDE-1 were used in this study.To monitor the composition of the intestinal microbiota of in situ hybridization (smFISH) were performed to verify Orsay virus infection of C. elegans. rde-1 (ne219) mutant animals were more sensitive to infection with Orsay virus than were N2 animals, as measured by viral load . Previous work suggested that C. elegans intestinal cells are structurally altered by Orsay virus infection and the viral particles are mainly localized to intestinal cells (rde-1 (ne219) mutant (rde-1_OrV) animals showed a much greater proportion of infected intestinal cells compared to N2 animals .Quantitative real-time polymerase chain reaction (qRT-PCR) and single molecular fluorescence al cells . Using sFigures 2, 3, P > 0.05). According to the weighted UniFrac distance measurements, a clear separation was found between uninfected N2 (N2_mock) animals and uninfected rde-1 (ne219) mutant (rde-1_mock) animals. The weighted UniFrac distance in N2_mock group was greater than in rde-1_mock group, and this demonstrated that the microbial diversity in N2 animals was significantly greater than in rde-1 (ne219) mutant animals. The variation of microbial characteristics was similar for rde-1 (ne219) mutant animals but significantly greater for N2 animals  animals showed no significant difference in intestinal microbial diversity compared with uninfected animals  mutant animals compared with N2 animals. Orsay virus-infected rde-1 (ne219) mutant animals had lower OTU richness than uninfected animals . The Shannon diversity indices showed that rde-1 (ne219) mutant animals tended to harbor fewer diverse bacterial communities than did N2 animals, and that this decrease in diversity was exacerbated by Orsay virus infection . The equitability index and Simpson index  indicated that the microbial communities of uninfected N2 animals exhibited the greatest diversity, and those of Orsay virus-infected rde-1 (ne219) mutant animals exhibited the lowest . Therefore, N2 animals exhibited higher levels of biodiversity and unevenness estimations compared to RDE-1-deficient animals. This indicated that RNAi plays a role in maintaining microbiomes during virus infection and the defective RNAi response in rde-1 (ne219) animals made the animals unable to maintain their microbiome when challenged with virus infection.We analyzed microbial diversity  to determine whether RDE-1 deficiency and Orsay virus infection were associated with the intestinal microflora of rde-1 mutant animals using LEfSe. The bacterial compositions differed between uninfected N2 and rde-1 (ne219) mutant animals. In the latter, the gut microbiome was significantly enriched in Actinobacteria, including Leucobacter and Pseudoclavibacter; Proteobacteria, including Acinetobacter and Burkholderia; and Alphaproteobacteria, specifically, Ochrobactrum . In contrast, the intestinal microbiome of uninfected N2 animals was enriched in Bacteroidetes, including Myroides and Sphingobacterium .To evaluate the response of the gut microbiome to RDE-1 deficiency and virus infection, we compared the microbial clades of infected and uninfected N2 and rde-1 (ne219) mutant animals. Orsay virus infection resulted in marked enrichment in the genera Sphingobacterium, Brevundimonas, and Trabulsiella . However, compared with Orsay virus-infected N2 animals, the relative abundances of Rhodococcus, Ochrobactrum, Tetrathiobacter, Delftia, Trabulsiella, and Acinetobacter were higher in rde-1_OrV animals , while the relative abundances of Pseudochrobactrum, Phyllobacterium, Agrobacterium, Shinella, and Stenotrophomonas were higher in N2_OrV animals . These results suggest that Orsay virus infection and RDE-1 deficiency exert marked effects on the composition of the gut microbiome.To identify the microbial taxa that respond to Orsay virus infection, we examined the microbiomes of infected and uninfected C. elegans after Orsay virus infection, we compared the mRNA profiles of mock and Orsay virus-infected N2 and rde-1 (ne219) animals.To assess the relationship between the intestinal microbiome and gene expression in rde-1 (ne219) mutant animals . We searched for genes that were significantly differently expressed  between uninfected and infected rde-1 (ne219) animals. A total of 144 genes were specifically induced or repressed by Orsay virus infection  animals . In contrast, genes downregulated in rde-1 (ne219) animals were mainly involved with lipid metabolism, lipid transport and fatty acid metabolism .The cluster analysis indicated that a large set of genes was associated with the difference between infected and uninfected C. elegans upon Orsay virus infection. Gut microbiome diversity was decreased in rde-1 (ne219) mutant animals, and several genes were associated with differences between infected and uninfected rde-1 (ne219) mutant animals. Our findings indicate that the RNAi pathway may play a role in maintaining the microbiome during virus infection.In this study, we evaluated the complexity of intestinal microbial communities in wildtype and innate immunity-deficient mutant rde-1 deficiency was associated with decreased microbial diversity, which was further reduced by Orsay virus infection. The gut microbiota status is a determining factor of host health. The intestinal microbial diversity of the rde-1 (ne219) mutant was further reduced, suggesting that the mutant animals were unstable and easily disturbed by external factors. Our results are consistent with a previous report of microbial enrichment and complexity in healthy hosts  mutant animals, Sphingobacterium was enriched in uninfected N2 animals, suggesting that Orsay virus infection and genetic factors influence the microbial community. Sphingobacterium is characterized by high concentrations of sphingophospholipid components , which facilitates bacterial competition, colonization, and survival within the host  mutant animals. Several genes were specifically induced or repressed by Orsay virus infection. acdh-1, a “dietary sensor” (rde-1 (ne219) mutant animals. This indicated that stress response genes were stimulated by virus infection and possibly associated with the immune response. In the presence of Orsay virus, genes related to the innate immune response, such as a cluster of C-type lectin (CLEC), were significantly up-regulated in the rde-1 (ne219) mutant animals. CLEC, which contribute to immune specificity in both vertebrates and invertebrates, maintain gut homeostasis by symbiotic microbiome offsets of the gut immunity in mosquitoes  mutant animals indicated that the CLEC genes may contribute to intestinal microbiome alternation associate with virus infection in C. elegans with defective immune response. Moreover, multiple genes were down-regulated in rde-1 (ne219) mutant animals, which highlights the role of sensor molecules in lipid metabolism, lipid transport and fatty acid metabolism, particularly vitellogenins (VITs). Lipid metabolism is an important pathway associated with aging  mutant animals indicates that VITs may be involved in the interactions among host genetic factors, virus infection and the microbiome.To investigate the changes in the intestinal microbiota community and the expression of related genes in  sensor” , was sigsquitoes . The gutsquitoes . The gensquitoes . The up-th aging . Besidesth aging . VITs weth aging . VITs in pathway . The dowC. elegans. This study reports for the first time the effect of Orsay virus infection on the intestinal microbiome and expression of related genes of N2 and rde-1 (ne219) mutant C. elegans. Our results demonstrate that intestinal microbiota diversity may be associated with host genes during virus infection. Consistent with a previous report that host genotype affects the intestinal microbiota, our data may help to explain human diseases caused by different genotypes associated with a variety of intestinal microbiome. Furthermore, our results may lead to a better understanding of the mechanism and evolution of the mutualistic relationship between a host and its gut microbial community based on nematode molecular genetics and immunity.The intestinal microbiota plays a stimulatory role in viral infection . HoweverYG contributed to design all the experiments, bench work and draft the manuscript. ZX contributed to analyze data. SC contributed to smFISH experiment. FC contributed to design all the experiments and draft the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China.A total of 78 bat liver samples  were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses.Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels.Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Orthohepevirus D of the family Hepeviridae.This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species  Nearly 60% of emerging infectious diseases in humans are zoonotic, with up to 70% of them being found to originate from wildlife . Bats haPrevious studies mainly focused on bat-borne viruses that are transmitted via respiratory droplets . HoweverHepatovirus in the family Picornaviridae. Hepatitis B virus (HBV) is classified as belonging to the genus Orthohepadnavirus in the family Hepadnaviridae. Hepatitis C virus (HCV) is classified as belonging to the genus Hepacivirus in the family Flaviriridae. Hepatitis D virus (HDV) is considered to be a subviral satellite because it can only propagate in the presence of HBV. Hepatitis E virus (HEV) is classified as belonging to the genus Orthohepevirus in the family Hepeviridae. Hepatovirus-related sequences have been identified in 13 species of bat collected in North America, Europe, and Africa [Hepatitis viruses include hepatitis viruses A, B, C, D, and E, which cause human hepatitis diseases. Hepatitis A virus (HAV) is classified as belonging to the genus d Africa . Hepadnad Africa , 8–10. Hd Africa . Hepevird Africa . These rThere are around 120 species of bat in China; however, only limited information has been reported regarding the hepatitis viruses, a novel Orthohepadnavirus in pomona roundleaf bats from Yunnan province was identified in 2015 . In thisCytB) gene following previously described methods [A total of 78 liver tissue samples were collected from dead bats caused by accident during sampling, which comprised two families, five genera, and 17 species, and used for virus screening Table . Differe methods .Table 2DHepatovirus, the polymerase gene of viruses in the family Hepadnaviridae, the RdRp gene of viruses in the genus Hepacivirus [Hepeviridae [RNA was extracted from tissue using the QIAamp Viral RNA Mini Kit  following manufacturer’s instructions, and cDNA was synthesized using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase . The extracted RNA from liver was tested by nested or heminested reverse transcription PCR (RT-PCR) using degenerate primers based on the conserved domain of the RNA-dependent RNA polymerase (RdRp) gene of viruses in the genus acivirus , and theeviridae  (Table 3The complete genomic sequences of one hepadnavirus strain and one hepevirus strain were amplified using PCR with degenerate primers (the primers are available upon request). The genome ends were amplified using a 5′-Full RACE Kit . The PCR products underwent gel purification with MinElute Gel Extraction Kit  and they were sequenced with both forward and reverse primers using the 3100 Sequencer. The sequencing chromatograms were inspected for overlapping multicolor peaks, which are an indicator of sequence heterogeneity in the amplicons. The PCR products were cloned using the pGEM-T Easy Vector System  and at least three clones for each PCR fragment were sequenced to obtain a consensus sequence.Hepadnaviridae and Hepeviridae used in the phylogenetic analyses are presented in Figs. The preliminary sequence management and analysis were carried out using Geneious version 9.1.3  and the sequence alignment and editing were performed using MAFFT . The phyVirus load of bat hepevirus and hepadnaviruese of different tissues was measured by using photometrically quantified in vitro RNA transcripts and specific real-time RT-PCR primers Table . QuantifAmong the 78 bat liver samples, four were positive for hepadnavirus from Jinning city, Yunnan province and only one was positive for hepevirus from Xianning city, Hubei province Fig. . HoweverR. sinicus (designated BtHBVRs3364 and BtHBVRs3366) and R. affinis (designated BtHBVRa4325 and BtHBVRa4328)  was determined and it was found to have a length of 3,272 nucleotides. The virus has an identical genomic organization to other hepadnaviruses, with four open reading frames (ORFs) encoding the surface (S), polymerase (P), core (C), and X proteins. In addition, the typical direct repeat (DR) sequences for viral genome replication and the secondary structure Ɛ–loops for viral reverse transcription were present in the BtHBVRs3364 genome. A detailed comparison of the full-length genomic sequence and the ORFs of the virus with other known hepadnaviruses is shown in Table The full-length genomic sequence of a sample from A phylogenetic tree was constructed based on the alignment of the full-length genomic sequence of BtHBVRs3364 with those of representative hepadnavirus strains available in GenBank. As shown in Fig. M. davidii, from Hubei province was positive for hepevirus . The genomic sequence of BtHEVMd2350 was found to have a length of 6,607 nucleotides (excluding the poly(A) tail at the 3′ end). This is slightly shorter than BS7 , the only reported bat hepevirus with a fully sequenced genome, which was identified from the serotine bat, Eptesicus serotinus, in Germany [Hepeviridae were also found in BtHEVMd2350: ORF1 encodes a nonstructural polyprotein that includes the RdRp, ORF2 encodes the capsid protein, and ORF3 encodes a multifunctional protein. Notably, most of the elements and domains characterized in BS7 could be found in BtHEVMd2350, but with a high level of divergence  and followed by spleen (7.3 × 108 RNA copies per gram of tissue), intestine and kidney, but not detectable in the brain. For bat hepadnavirus, the highest viral load was found in the liver of BtHBVRs3364 (2.0 × 1010 RNA copies per gram of tissue), the virus load of tissues of BtHBVRs3366, BtHBVRa4325, and BtHBVRa4328 were relatively similar .Viral load detected by qPCR in different tissues were presented in the Fig. M. davidii in Xianning city, Hubei province. The full-length genomic sequences of one of the two hepadnaviruses from R. sinicus and the hepevirus from M. davidii were determined.Since the discovery of genetically diverse hepatitis virus-related sequences in bats, bats have been considered to be important natural reservoirs for hepatitis viruses, and potential sources of human diseases . HoweverThe phylogenetic analysis indicates that the bat hepadnavirus found in this study is closely related to roundleaf bat hepadnaviruses, which were discovered in Pu’er city, Yunnan province in 2011 , but shoHepeviridae. According to the latest proposal of the ICTV in 2016, amino acid distances of concatenated ORF1 and ORF2 (lacking hypervariable regions) greater than 0.088 could then act as threshold to demarcate intra- and inter- genotype distances [M. davidii, and that found in the German serotine bat, E. serotinus (the only reported bat hepevirus with a full-length genome) shared significant diversity from both nucleotide and amino acid levels, we propose that they can be grouped into the species Orthohepevirus D which is divided into two genotypes: D1 and D2.Regarding the bat hepevirus, the phylogenetic analysis indicates that the known bat hepeviruses are highly divergent from other mammalian hepeviruses and that they form an independent branch in the family istances . The hepOur results provide further evidence to support the theory regarding the long-term co-evolution of hepadnaviruses and hepeviruses with their hosts, and the theory that bats act as major natural reservoirs for these hepatitis viruses. Our results have limitations due to the small sample size used, which was a result of the protection of bat populations in China, as bats play important roles in the pollination of plants and in pest control, as they feed on insects. However, based on our discovery of hepatitis viruses in bats, it is expected that there are many more hepatitis viruses circulating in numerous bat species and in various geographic regions. In order to obtain larger sample sizes, non-invasive methods of virus detection should be considered for future studies.


Calculations show that total virus turnover was fast with 2 to 4 days, whereas virus-mediated cell turnover was calculated to range between 5–13 or 33–91 days depending on the assumed burst sizes (number of viruses released upon cell lysis) of 14 or 100 viruses, respectively. During the experiments, the homogenized sediments in the reactors became vertically structured with decreasing microbial activities and increasing impact of viruses on prokaryotic mortality with depth. Tidal simulation clearly showed a strong accumulation of viruses and cells in the top sections of the reactors when the flow was halted indicating a consistently high virus production during low tide. In conclusion, cell lysis products due to virus production may fuel microbial communities in the absence of advection-driven nutrient input, but are eventually washed off the surface sediment during high tide and being transported into deeper sediment layers or into the water column together with the produced viruses.Sandy surface sediments of tidal flats exhibit high microbial activity due to the fast and deep-reaching transport of oxygen and nutrients by porewater advection. On the other hand during low tide, limited transport results in nutrient and oxygen depletion concomitant to the accumulation of microbial metabolites. This study represents the first attempt to use flow-through reactors to investigate virus production, virus transport and the impact of tides and season in permeable sediments. The reactors were filled with intertidal sands of two sites (North beach site and backbarrier sand flat of Spiekeroog island in the German Wadden Sea) to best simulate advective porewater transport through the sediments. Virus and cell release along with oxygen consumption were measured in the effluents of reactors during continuous flow of water through the sediments as well as in tidal simulation experiments where alternating cycles with and without water flow (each for 6 h) were operated. The results showed net rates of virus production (0.3–13.2 × 10 Approximately 20% of the primary production in the oceans takes place at the continental shelves Jahnke, . Due to In North Sea seafloor sediments, which are predominantly covered with sands . With this, we aimed to estimate virus and cell production rates including the influence of season and tidal cycles. We hypothesized a dependency of virus activity and abundance on prokaryotic activity and a dynamic interplay between viruses and host cells that is influenced by advection-driven transport during tidal cycles.Surface sediment samples were collected from the Janssand sand flat in June 2016, July 2016, February 2017 and August 2017 and additional samples from the Spiekeroog beach site in November 2016 Figure . During Seawater for the flow through the reactors was collected each time off site Janssand  and stored at 4°C in the dark. The water temperatures were 17°C in June 2016, 20°C in July 2016, 7°C in November 2016, 2°C in February 2017 and 19°C in August 2017.3 and 8 × 106 viruses ml−1 and cell numbers between 4 × 102 and 4 × 106 cells ml−1, because in some occasion cell growth occurred during water storage after the autoclaving and filtering of the seawater.The seawater was filtered through 0.45 μm and 0.2 μm filters and additionally autoclaved either before or after 0.2 μm filtration. A single 10 l-reservoir was covered with a black cloth to keep it dark and was regularly exchanged. During the experiments in February and August 2017, the seawater in the reservoir was additionally bubbled with air. Virus and cell counts in the reservoir were monitored in detail only for the experiment in February 2017. Virus numbers ranged between 8 × 10−1. For reasons of practicability and availability of facilities, the flow-through reactors and the seawater were kept at 15°C in the dark for all experiments, which represents the annual average temperature.Flow-through reactors made of acrylic glass with a height of 17 cm and a diameter of 4.2 cm were filled with seawater to which homogenized surface sediment was slowly added avoiding the trapping of air bubbles , instead in these control reactors water was continuously pumped through the sediment. With the beach sediment, one reactor at the end of the experiment was used as a dead control. For this, seawater with 2% formaldehyde was pumped for 30 h through the reactor.Samples for virus and cells counts in the effluent were taken twice a day during continuous flow. For tidal simulation experiments, selected tidal cycles were sampled in high frequency directly after the restart of the water flow. For enumeration of viruses and cells in the effluents, 1 ml of effluent was collected from the outflow port into a reaction vessel, fixed with 20 μl of 25% glutardialdehyde and frozen at −80°C. Similarly, samples from the reservoir were taken regularly. For oxygen measurements in the effluent, an oxygen flow-through cell with an integrated contactless optical oxygen sensor  was attached to the outflow port. Additionally, oxygen concentration of the seawater reservoir was regularly measured with the flow-through cells attached behind the peristatic pump.3 of sediment was fixed with 1 ml of 4% formaldehyde and stored at 4°C. For virus counts, 1 cm3 of non-fixed sediment was immediately frozen at −80°C. Porosity was calculated from wet weights and dry sediment densities in triplicates.For enumeration of viruses and cells in the sediment, samples were collected from homogenized sediments before the flow-through reactors were filled as well as at the end of each experiment from the reactors. For cell counts, 0.5 cm3) were centrifuged for 3 min at 13,000 × g and the supernatant was discarded. Cells were extracted with 1 ml 5 mM sodium pyrophosphate buffer for 15 min in the dark, followed by sonication for 3 min. Sediment particles were allowed to settle for 20 s and the supernatant was transferred into a new tube. Similarly, the sediment was washed 6–8 times with 1 ml PBS and the supernatants were pooled. The extract was filtered onto a 0.2 μm pore size filter  and stained with SYBR Green I. Cells were counted with an epifluorescence microscope Olympus bx51 within 10 to 20 randomly chosen counting fields and at least 400 cells counted.For the enumeration of prokaryotic cells from formaldehyde-fixed sediment, samples . Cells were counted by fluorescence microscopy and viruses were counted by flow cytometry as described below. The fractions of free cells and viruses in the porewater were calculated in comparison to the cell and virus numbers of bulk sediment extractions, respectively.For enumeration of viruses and cells in the effluent of the reactors, samples were thawed and viruses and cells counted by flow cytometry with a flow cytometer BD Accuri C6  according to Brussaard et al. . BrieflyCfree) and viruses (Vfree) were calculated based on the counts of viruses (VDCPW) and cells (CDCPW) per porewater volume, the porosity (ϕ) and cell (CDCbulk) and virus (VDCbulk) counts of bulk sediment extractions, respectively.The fractions of free cells (rt), with rt = V ϕ f−1 determined by the sediment volume (V), the porosity (ϕ) and the flow rate (f). The calculations include an average for virus counts (VDCefflux) and cell counts (CDCefflux) of the effluent, where counts from reactors that were sampled immediately after the restart of the water flow during tidal cycles as well as numbers at the start of the experiments when virus and cell numbers in the effluent had not stabilized yet (0.5–4.5 days for the respective experiments) were excluded. Net production rates of viruses (VPnet) and cells (CPnet) were calculated based on the following equations:Virus and cell production rates were calculated based on respective virus or cell numbers in the effluents, the retention time (Cin) and outflow (Cout) and the retention time (rt). For the oxygen consumption rates, the same time intervals than for the net virus and cell production rates were used. Oxygen consumption rates (OCR) were calculated according to the following equation:Oxygen consumption rates were calculated from the difference between oxygen concentrations in the inflow (VPgross) and cells (CPgross) were calculated from the net production rates based on the fraction of free viruses (Vfree) and cells (Cfree):The gross production rates of viruses (VPgross) and cells (CPgross) and the virus counts (VDCbulk) and cell counts (CDCbulk) in the sediment, turnover times of virus (VT) and prokaryotic communities (CT) (replacement of the standing stock of virus and cell numbers in the sediment) were calculated according to the following equations:Based on the gross production rates of viruses (VMPT) is a theoretical estimate on the prokaryotic turnover with the assumption that viral lysis is the sole mortality factor and was calculated based on the cell counts (CDCbulk) in the sediment, the gross virus production rate (VPgross) and the burst size (BS = the number of viruses released per cell lysis event):The virus-mediated prokaryotic turnover (VIPM) determines the proportion of the total prokaryotic mortality that is caused by virus lysis and was calculated according to the following equation  was used and is justified by different sample sizes and an unequal variance  and subsequently restarted to continuous flow for 6 h. Out of 12 successive tidal cycles conducted during the February experiment, four cycles were sampled immediately when the water flow was restarted after low tide. Maximum virus and cell numbers in the effluents were detected after the restart of water flow Figures  and reprSimilar increases in virus and cell numbers directly after the restart of the water flow during tidal simulations were found in all experiments  compared to February. In sediments from Spiekeroog beach, cell numbers were 6- to 40-fold lower Table . Virus c6 viruses ml−1 and 1.1 × 105 cells ml−1 to 1.2 × 104 viruses ml−1 and cell counts below detection, while the oxygen concentration in the effluent increased rapidly to a constant level of 93% compared to the reservoir with seawater  as well as oxygen consumption in the effluents of reactors with longer sediment columns , while virus numbers were significantly lower for the shortest columns in comparison to the longest columns (p < 0.01). For the August experiment, the four different column lengths showed significant differences for the oxygen consumption (p < 0.01), while for virus numbers the short, medium and long columns were significantly different and for cell numbers only the shortest column differed significantly from the other three columns (p < 0.01). In contrast by calculating average rates, oxygen consumption rates (μmol l−1 h−1) and net virus and cell production rates (cm−3 h−1) were higher for shorter sediment columns, i.e., smaller sediment volumes  (Hewson and Fuhrman, Virus production rates are commonly measured in incubations of undiluted or diluted sediment, where the increase in virus counts is quantified over several hours (Rastelli et al., −1 Table . Taking t Figure . Net andVirus production, cell production and oxygen consumption rates of all experiments were positively correlated Figure . Oxygen The flow-through reactors were started with homogenized sediments. Unexpectedly, highest average virus and cell production rates were determined for the short sediment columns compared to longer columns for the February and August experiments Table . We conc Figures .The differences of microbial activity in short and long columns and accordingly surface and bottom of the sediment columns were also observed during the tidal simulation experiments. Here, the maxima of virus and cell numbers were determined in the effluent that had been present as porewater in the surface sediment of each reactor during low tide Figures , 5C,D, aThe virus turnover in sediments was 1.8, 2.4, and 3.6 days for the top, middle and bottom sections of the columns for the February experiment, respectively Figure . Similarin situ sampling of surface sediments during low tide (Montanié et al., Permeable sandy sediments allow for advective transport through their pore spaces. In intertidal sediments during high tide, advection is mainly driven by pressure gradients formed by tidal and wave pumping. This leads to rapid “skin circulation” through the surface enhancing the transport of particulate and dissolved organic matter, other nutrients and electron acceptors like oxygen several cm down into the sediments (Jansen et al., VV and TE: designed the study and wrote the manuscript; VV, LS, and TE: performed the laboratory work and data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This costing study in Malawi is a first evaluation of a Maternal Influenza Immunization Program Costing Tool (Costing Tool) for maternal immunization. The tool was designed to help low- and middle-income countries plan for maternal influenza immunization programs that differ from infant vaccination programs because of differences in the target population and potential differences in delivery strategy or venue.This analysis examines the incremental costs of a prospective seasonal maternal influenza immunization program that is added to a successful routine childhood immunization and antenatal care program. The Costing Tool estimates financial and economic costs for different vaccine delivery scenarios for each of the major components of the expanded immunization program.In our base scenario, which specifies a donated single dose pre-filled vaccine formulation, the total financial cost of a program that would reach 2.3 million women is approximately $1.2 million over five years. The economic cost of the program, including the donated vaccine, is $10.4 million over the same period. The financial and economic costs per immunized pregnancy are $0.52 and $4.58, respectively. Other scenarios examine lower vaccine uptake, reaching 1.2 million women, and a vaccine purchased at $2.80 per dose with an alternative presentation.This study estimates the financial and economic costs associated with a prospective maternal influenza immunization program in a low-income country. In some scenarios, the incremental delivery cost of a maternal influenza immunization program may be as low as some estimates of childhood vaccination programs, assuming the routine childhood immunization and antenatal care systems are capable of serving as the platform for an additional vaccination program. However, purchasing influenza vaccines at the prices assumed in this analysis, instead of having them donated, is likely to be challenging for lower-income countries. This result should be considered as a starting point to understanding the costs of maternal immunization programs in low- and middle-income countries. Beginning in 2012, the World Health Organization (WHO) recommended that countries prioritize pregnant women above other high risk groups for receipt of influenza vaccine ,4. To daNational and international decision makers must consider both health and economic information, including program costs, when considering new vaccine introduction ,6. ThereThe purpose of this report is to describe the first evaluation of a Maternal Influenza Immunization Program Costing Tool (Costing Tool) for maternal immunization which was undertaken in Malawi. Malawi was selected for this pilot study due to its success in eliminating maternal and neonatal tetanus and to leverage related maternal influenza immunization feasibility studies that were being undertaken in-country. This is to our knowledge, the first costing study of maternal influenza immunization in a developing country.The Costing Tool is a Microsoft Excel-based micro-costing tool commissioned by WHO that estimates the incremental  resources required to add maternal influenza vaccination to an existing national immunization program over a five-year period. It was developed as part of a series of economic tools to inform decision making and to plan the introduction of a maternal influenza immunization program –14. The The Costing Tool estimates costs for each of the major components of the immunization program as illustrated in In addition to these categories, the Costing Tool estimates initial investment costs which are upfront, non-annualized costs that include introduction costs with the addition of cold chain and other capital costs over the five-year time horizon of the Costing Tool. It is important to note that the Costing Tool annualizes introduction, cold chain, and other capital costs across the user defined “useful life years” but these are not annualized in the initial investment costs. However, if the useful life of a capital good exceeds the five-year horizon of the tool, only the share of the capital cost included in the five-year horizon will be captured. As such, initial investment costs are the best representation of the up-front cost of the vaccination program.Across all of these categories, the Costing Tool utilizes and reports both financial and economic costs. Financial costs represent the value of resources purchased by the buyer such as the Ministry of Health (MOH). Financial costs are likely to include items such as the purchase of injection supplies, outreach allowances, and personnel per diems as well as purchased resources used in training and developing new communication materials. Economic costs represent the value of all resources used by the program regardless of who pays for those resources. Economic costs include all financial costs and are also likely to include items such as salaries of current health personnel, volunteer labor, donated supplies, and the opportunity cost of capital goods. The Costing Tool user guide contains a more comprehensive list of potential financial and economic costs by vaccination activity . The useIn addition to cost projections reported using the categories described above, the tool also reports several other cost metrics. The tool reports the number of immunized pregnant women on an annual basis, annual costs, and the cost per immunized woman across the time horizon of the tool. In this report, costs are reported in 2015 US Dollars, though the Costing Tool accommodates inputs and results in local currency or US Dollars for other reporting years.The Costing Tool is designed to be adapted to the local context and incorporate country characteristics including demographics, administrative levels, and vaccination strategies . While it is important to adapt the tool to the local context, the tool generates the best results when local experts and an experienced facilitator collaborate to conduct the costing analysis.In June 2015, the study team collected primary and secondary data on the costs of introducing a prospective maternal influenza immunization program after Malawi’s National Health Sciences Research Committee (NHSRC) provided Institutional Review Board approval for the study. PATH IRB deferred to NHSRC.Qualitative and quantitative data were collected from primary and secondary sources at the national, zonal/regional, district, and facility levels. Key informant interviews were conducted at the National Expanded Program on Immunization (EPI) and Health Education Unit. Interviews were also conducted with program managers and at two health centers in each of three districts: Dowa, Rumphi and Zomba. Additional information was collected from follow up questions with the National EPI Logistician, the officer in charge of procurement at UNICEF Malawi, and the Infrastructure Development Planning Officer at the Ministry of Health. Information from secondary data sources  also supplemented the interviews.Quantitative data on supply quantities, remuneration for staff time, expenditures, and other relevant expenses associated with vaccinations were gathered from record books at the national, district, and facility levels. These data were complemented by expert opinion in areas where documented information on the implementation of seasonal influenza vaccine was not available. Data collected included all costs linked to planning, training sessions at all administrative levels, social mobilization events, monitoring and supervision activities, procurement, storage of vaccines and supplies, immunization service delivery in static and outreach outlets, and eventual evaluation of the program.The Costing Tool pilot in Malawi estimates the incremental cost of introducing a prospective maternal influenza vaccine. We assume delivery would be based on existing pediatric and ANC immunization infrastructure. This assumes a strong EPI and ANC program and coordination between the two programs. This study estimates both financial and economic costs from the government perspective over a five-year period beginning in 2018. Costs were collected in US Dollars and Malawian Kwacha. Kwacha were converted to US Dollars based on average UN Treasury Operational Rates between June 2014 and May 2015, i.e. the year prior to data collection. A start year of 2018 was selected as it represents costs within the relatively near term but would still allow a couple of years of preparation, if an introduction decision were made. For the purposes of this costing study, we assume Malawi would introduce seasonal maternal influenza immunization across all districts in the first year. Cost components are those illustrated in We consider women who attend one ANC visits as a optimistic assumption for those that may receive vaccination in a low-income country setting. We begin with 95% of women who have at least one ANC visit as indicated by Malawi’s 2010 Demographic Health Survey and apply a uniform dropout rate between ANC visits one, two, three, and four . We adjuThe following section describes our base scenario results and then discusses alternative scenarios. Alternative scenarios include a lower ANC coverage scenario as well as a low and high coverage scenario in which Malawi would purchase the vaccine in a 10 dose vial.The base scenario financial cost of the maternal immunization program is approximately $1.2 million over five years. Overall financial and economic costs are displayed in Over the five-year time period, introduction costs account for approximately 70% of financial costs with recurrent costs accounting for approximately 30%. Introduction costs include microplanning, training and social mobilization/information, education, and communication (IEC) at 14%, 13%, and 7% respectively. Capital costs, i.e. cold chain, account for 34% of total financial costs. Among recurrent costs, service delivery and supervision account for 16% and 10% of total costs respectively. The bulk of service delivery costs are associated with outreach (i.e. per diem and transport). Continuing IEC contributes 5% of total costs. Vaccines and supplies are only a small component of financial costs as this scenario assumes a donated vaccine; other recurrent costs are minimal.We now examine the annual costs of the program over five years. Microplanning, training, and social mobilization are the largest categories while cold chain, service delivery, and supervision are smaller cost components. In the ensuing years, cold chain, service delivery, and supervision are the main costs but social mobilization adds costs every two years. Notably, vaccines and supplies are only a small financial cost since we assume a donated vaccine.From an economic cost perspective , the cosOver the initial five years of the immunization program, 2.3 million pregnant women would be reached under our base scenario assumptions. Over this period, the cost per immunized pregnancy is estimated at $0.52  and $4.58 (economic). Immunization coverage is 47% of the eligible population.The base scenario described above represents the costs associated with a potential maternal influenza immunization program in a low-income country. We now examine a scenario with a less optimistic ANC coverage and then examine the costs of a maternal influenza immunization program with a purchased vaccine and different presentation, under both low and higher coverage scenarios.The second scenario replicates the base scenario but decreases vaccine coverage. This scenario assumes that women with at least four ANC visits will be eligible to receive influenza vaccine if they attend during the vaccination season. It starts with 46% of women who have at least four ANC visits as indicated by Malawi’s 2010 Demographic Health Survey but assume no dropoutsbetween ANC visits one, two, three, and four . We agaiOverall financial and economic costs decrease to $1.0 and $6.3 million, respectively. The financial and economic cost per immunized pregnant woman falls to $0.79 and $5.06, respectively. The higherer cost per immunized woman is due to spreading fixed costs over 1.1 million fewer immunized pregnant women. Immunization coverage is 26% of the eligible population. Additional detail on this scenario can be found in Table A in This scenario considers only women that attend four or more ANC visits as the potential beneficiaries. Malawi purchases a vaccine rather than benefits from a donation program. Since no low- or lower-middle income countries reported purchasing a 10 dose vial of seasonal influenza vaccine in the WHO Vaccine Product, Price and Procurement Database, we utilized the lowest reported price ($2.80) for a 10 dose vial reported by Pan American Health Organization . The chaPurchasing a vaccine greatly increases the financial cost of vaccines and supplies but reduces cold chain storage requirements and costs see  below. AThis scenario returns to high vaccine coverage with 95% of women who having at least one ANC visit and a uniform dropout rate between ANC visits. The overall cost of the program increases to $13.5 million over five years, primarily due to vaccine and supply costs. The program reaches 2.3 million women at a financial (economic) cost per immunized pregnant woman of $5.95 ($6.24). Immunization coverage is 47% of the eligible population. Annual program costs are available in In this costing study of a maternal influenza immunization program in Malawi, base scenario financial and economic costs per dose delivered are approximately $0.50 and $4.50 respectively. Introduction costs for our base scenario account for 70% of total financial costs across the five-year period and recurrent costs are the remaining 30% of total financial costs. Microplanning, training, and cold chain are the largest components of introduction costs. Service delivery is the largest component of recurrent costs at 16% of total financial costs. Relative to some new vaccine introductions such as HPV, costs associated with social mobilization are slightly lower but in the same range. For example, Hutubessy et al. (2012) find that social mobilization and IEC account for 10% of the financial costs . Our basOur base scenario includes vaccine costs only as an economic cost. With this in mind, our base scenario financial cost per dose roughly corresponds to recent multicountry analyses of immunization delivery costs ,36. AverNoting the assumptions made in our base scenario, delivery costs do not seem overwhelming. However, if Malawi or another low-income country were to consider purchasing a vaccine, this would change the financial outlook. For example, purchasing a vaccine at the lowest prices reported by a low- or lower- middle income country increases the financial costs per dose to between $5 and $7 (depending on the scenario) and would have dramatic budget implications. While a full assessment of affordability is beyond the scope of this costing analysis, we note that Malawi’s per capita total health expenditure is $26 per year meaning that the budget impact of a maternal influenza immunization program would be large . Each opProgram costs and benefits are not the only consideration . IntroduThere are several limitations to this study that deserve mention. First, this study was used to pilot a new tool that is not yet WHO endorsed. All results should be considered in relation to current knowledge and future piloting. In addition, both data collection and analysis were affected by the uncertainty surrounding a prospective immunization program so our interviews with EPI program staff were not informed by active maternal immunization program planning. This analysis also assumes many activities are built upon a well-functioning system so a number of activities would not incur incremental costs . While we believe our estimates are realistic, they are only reflective of the delivery strategy and our knowledge of the country context. If a country selected a strategy that required more stand-alone outreach or supplemental activities, costs would increase. The Costing Tool does not allow for uncertainty analysis outside of examining alternative scenarios so these scenarios should be interpreted carefully. Finally, the costs of EPI/ANC program coordination are not included in these estimates and would increase the costs of the program slightly.This study demonstrates the successful use of a new influenza vaccine costing tool and estimates the financial and economic costs associated with a prospective maternal influenza immunization program in a low-income country. The information gained through this study not only helps inform our understanding of costs in Malawi, but may also benefit other countries as little is known of the costs of maternal immunization programs. Our analysis estimates that the incremental cost of a maternal influenza immunization program integrated into the ANC platform does not differ dramatically from the incremental costs one might expect if adding another antigen to a childhood vaccination program on a cost per dose basis. Maternal immunization delivery costs may be reasonable assuming the EPI and ANC system is capable of serving as the platform for an additional vaccination program. However, purchasing influenza vaccines at the prices assumed in this analysis is likely to be challenging for lower-income countries. These conclusions should be viewed as the product of one of the first studies of its kind and should be interpreted cautiously until further corroboration. WHO will conduct multiple iterations of piloting and refinement of the tool with eventual plans for a formal review by immunization economics experts and endorsement by WHO IVIR-AC before the tool is made available in the public domain. While we believe our results accurately reflect costs under the scenarios described, these findings should be corroborated by similar studies in other countries to confirm their generalizability and applicability to the local context prior to being used for policy decisions.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.