The susceptibility of sheep, cattle, pigs, chickens and chicken embryos to Zika virus infection was evaluated by experimental inoculation with Zika virus Thailand strain isolated from a Canadian traveler in 2013. The inoculated animals did not develop any clinical signs of disease nor evidence of Zika virus replication in peripheral blood, cerebrospinal fluid and tissues including brain and spinal cord assessed by real-time RT-PCR. Sera were also negative for Zika virus antibodies by Zika virus neutralization assays as well as Zika virus immunoperoxidase staining of Zika infected Vero cells. Chicken embryos were inoculated by different routes including yolk sac (4 day old embryos), chorioallantoic membrane (8 day old embryos), amniotic fluid (8 day old embryos) and intravenous routes (12 day old embryos). Virus replication in chicken embryos was observed in the brain and body tissues following intravenous (IV), yolk sac (YS), chorioallantoic membrane (CAM), and amniotic fluid (AF) inoculation routes. The highest mortality was observed in embryos inoculated via yolk sac. The dead embryos showed diffuse muscular hemorrhages. The yolk sac inoculated chicken embryos showed delayed hatching and displayed neurological signs immediately after hatching. These studies demonstrate that 8 week old sheep, 6 month old cattle, 4 week old pigs, and 4 week old chickens are not susceptible to Zika virus infection when inoculated experimentally and therefore unlikely to pose a risk as Zika virus reservoirs. However, chicken embryos are highly susceptible to Zika virus resulting in clinical disease of chicks after hatching. This study demonstrates that Zika virus has a tropism for embryonic tissue and that chicken embryos can be used as a model to study Zika virus replication and pathogenesis. Aedes aegypti, a mosquito that can also transmit dengue fever, chikungunya and yellow fever viruses. In recent outbreaks in humans, Zika virus caused a mild self-limiting infection with clinical signs of fever, rash, conjunctivitis, arthralgia, and arthritis during an outbreak on Yap Island of the Federated States of Micronesia in 2007  and intravenous inoculation of 1.0 ml with Zika virus Thailand (6 TCID50/ml propagated in Vero cells. Chickens were given five subcutaneous inoculations 0.1 ml each and 0.5 ml intravenous inoculation of Zika virus Thailand (106 TCID50/ml). All animal experiments were conducted under the approval of the Canadian Science Centre for Human and Animal Health Animal Care Committee, which follows the guidelines of the Canadian Council on Animal Care. All animals were observed twice daily, with clinical signs recorded throughout the study. Rectal temperatures were measured prior to inoculation for baseline levels and daily from 1 to 21 days post-infection (dpi). Blood and sera, were collected from sheep on days 1, 2, 3, 6, 8, 10, 13, and 21 post-inoculation. Blood and sera were collected from cattle on days 1, 3, 6, 8, 10, 13, and 20 post-inoculation. Blood and sera were collected from pigs on days 3, 5, 6, 7, 10, 12, 14, and 21 post-inoculation. Blood and sera were collected from chickens on days 3, 4, 6, 10, 13, 17, and 21 post-inoculation. Three weeks after inoculation, sheep, cattle, pigs, and chickens were euthanized and necropsies were performed. During the necropsy, animals were assessed for gross pathology cerebrospinal fluid (CSF) as well as tissues including the brain  and spinal cord were collected during the necropsies.Six (8 week old) Rideau Arcott sheep, four (6 months old) Holstein calves, and four (4 week old) Landrace/Large White cross piglets were obtained from a high health status herd operated by a recognized commercial supplier in Manitoba, Canada. Six (2–4 week old) Leghorn chickens were obtained from a specific pathogen free status flock operated by CFIA Ottawa. The age of animals selected was based on availability to procure animals of the youngest age which were not neonates. All animals were housed in separate Biosafety Level 3 animal cubicles at the National Centre for Foreign Animal Disease , and were fed a complete balanced diet and water Thailand  at 106 T7 PFU/ml, or as 10-fold dilutions. The inoculation procedures were done as follows. For intravenous (IV) inoculations 12 day old embryonated chicken eggs (ECEs) were used. For yolk sac (YS) inoculations 4 day old ECEs were used and for chorioallantoic membrane (CAM) and amniotic fluid (AF) inoculation 8 day old ECEs were used.Zika virus Thailand was inoculated into fertilized specific pathogen free (SPF) chicken eggs  at different embryonic stages using different routes of inoculations. The virus was administered using 100 μl inoculations at various doses with the highest doses of 10Prior to inoculations, eggs were allowed to cool at room temperature for approximately 1 h. For IV inoculation, 27 gauge hypodermic needles were used. For all other routes 1½ inch 23 gauge needles were used. Once inoculated the eggs were incubated at 33°C in a nonrocking incubator for 24 h with 55% relative humidity (RH), and transferred to a nonrocking incubator set at 37°C with 55% RH. Eggs were candled twice daily. All eggs dying within the first 24 h were discarded. The dead eggs were stored at 4°C a minimum of 1 h before the tissues  were harvested. From small embryos, all organs were pooled as body parts.4 PFU/ml and 36 control eggs were inoculated with sterile phosphate buffered saline (PBS). Eggs were incubated for 7 days in the egg incubator in the laboratory and transferred to a table top hatching incubator with turning trays located in animal pens.Two hatching experiments were conducted, one via CAM inoculation and the second via YS inoculation. For CAM inoculation, a group of thirty six 8 day-old embryonated eggs were inoculated with Zika virus dose of 104 PFU/ml, and 20 eggs with sterile PBS. The eggs were incubated for 11 days in the egg incubator and transferred to a table top hatching incubator in the animal pens.For the yolk sac inoculation, a total of 50 embryonated eggs were used. Zika virus Thailand was inoculated into 30 eggs via yolk sac on day 4 using a dose of 10Following inoculation, all eggs were assessed for viability by candling twice daily, and were allowed to hatch. Hatchability was determined and hatched chicks were evaluated for potential birth defects and abnormal clinical signs.Total RNA was extracted from allantoic fluid, yolk, whole blood and 10% tissue homogenates using MagMAx 5X Pathogen DNA/RNA kit Extraction Kit following the manufactures protocol . Two independent Zika real-time RT-PCR assays were performed to detect Zika virus RNA in the samples. Assay #1  detects 2 for 4 days and a second overlay was applied containing 1% Noble agar and 0.02% neutral red vital stain (Sigma) in MEM (2x). Plates were incubated at 37°C with 5% CO2 for an additional 3 days. Zika virus plaques were counted using a white light of a transilluminator (Fisher Scientific).Sera were assessed for Zika virus neutralization activity using Vero cells. Animal sera from DPI 0 and DPI 21 post-inoculation were diluted 10-fold in BA-1 diluent and mixed with 200 focus-forming units of Zika virus. The virus-sera mixtures were incubated for 1 h at 37°C and transferred onto a monolayer of Vero cells in a 24-well plate at 37°C. After a 1 h incubation, the monolayers containing the virus-sera mixture were overlaid with MEM (2x)  containing 4% fetal bovine serum and 1% Noble Agar . The plates were incubated at 37°C with 5% CO2. The medium was removed and cells were washed three times with phosphate-buffered saline (PBS)-Tween (200 ml/well) (PBST). The cells were then fixed with 10% neutral-buffered formalin in PBS (200 ml/well) for overnight at 4°C. The fixative was removed, and the cells were washed 3 times with PBS-T (200 ml/well) and blocked with blocking buffer (Sigma) for 1 h at 37°C. The blocking buffer was removed, and wells were washed three times with PBST. Sera were diluted 10-fold in PBS and 100 μl of diluted sera was added to the fixed cells and incubated for 1 h at 37°C. The primary antibody was removed and wells were washed three times with PBST. One hundred microliters of HRP-rec- Protein G antibody (Life Technologies) for sheep, pig, calf and donkey anti-chicken IgG (Jackson ImmunoResearch Laboratories) diluted 1:1,000 in PBS was added to each well for 1 h at 37°C. Plates were washed again three times with PBST and TrueBlue peroxidase substrate  was added. After development, the substrate was removed and the plates were rinsed with water and dried. Plates were assessed for immunostaining by microscopy.Immunoperoxidase staining: Presence of Zika virus specific antibodies in serum collected from infected animals were assessed by immune-peroxidase staining. Vero cells were infected with Zika virus using MOIs of 0.1, 0.01, 0.001 and incubated for 5 days at 37°C with 5% COFollowing inoculation with Zika virus, sheep, cattle, pigs, and chickens did not develop any clinical signs. All infected animals were clinically normal, had physiological body temperatures throughout the study, and displayed no change in appetite. Whole blood collected at various time points from sheep, cattle, pigs and chickens was assessed for the presence Zika virus genomic material using two different real-time RT-PCR assays. Zika virus RNA was not detected in any blood sample collected at any time point following Zika virus inoculation. Three weeks after inoculation the animals were euthanized and necropsies were performed. There was no gross pathology observed during the necropsy and neural tissues  from the sheep, cattle, pigs and chickens were negative for Zika virus RNA. Sheep, cattle, pigs, and chickens did not develop any detectable antibodies specific for Zika virus at any time points following inoculation by virus neutralization testing and immunoperoxidase staining.Zika virus Thailand strain inoculated by four different routes including IV, CAM, AF, YS were lethal to chicken embryos . YS inoc5 titer resulted in 60% survival by 5 dpi and 106 titer resulted in 40% survival by 6 dpi. Doses 104 or less resulted in no mortality in inoculated embryos. Eggs inoculated with 104 Zika virus via YS route showed 20% mortality by 5 dpi, and 80% of the embryos survived  showed depression, ataxia and was sitting on hocks. Another chick (#4) was mildly depressed and ataxic . On day In eggs inoculated with Zika via CAM route showed no significant differences in hatchability and health of the chicks compared to the PBS controls. The hatchability of YS inoculated embryos was lower with only 50% eggs hatching compared to control and CAM inoculated embryos which showed over 80% hatchability although not significantly significant .It was recently demonstrated that pig cell line PK-15 and chicken cell line DF-1 were able to support Zika virus replication . The abiThe lack of viremia observed in sheep, cattle, pigs, and chickens demonstrate that these animals are not likely to serve as reservoirs for Zika virus. It was recently demonstrated that both rhesus and cynomologus macaques are highly susceptible to Zika infection under experimental conditions  and thatIt has been reported that a small percentage of cows, sheep, goats and chickens had Zika specific antibodies in sero surveillance studies , 9. HoweEven though these animal species were not susceptible to Zika virus infection, this does not mean that embryos of these species are not susceptible . It has A. albopictus, which will feed on these species, although further evaluation of these species as sentinels is required. The use of birds as a sentinel species as a surveillance tool for West Nile virus has been previously demonstrated (The results from this study confirms the study by Ragan et al.  that farnstrated  and is onstrated . The resAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Canadian Science Centre for Human and Animal Health Animal Care Committee.AA, TT, CC-B, YB, VG, UK, DS, and SB contributed conception and design of the study. AA, TT, CC-B, YB, DS, and SB performed the experimental work. AA and SB wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


Background: The objective is to study previously unexplored trends of birth hospitalization and readmission costs for late preterm infants (LPIs) in the United States between 2005 and 2016. Methods: We conducted a retrospective analysis of claims data to study healthcare costs of birth hospitalization and readmissions for LPIs compared to term infants (TIs) using a large private insurance database. We used a generalized linear regression model to study birth hospitalization and readmission costs. Results: A total of 2,123,143 infants were examined . The proportion of LPIs requiring readmission was 4.2% compared to 2.1% of TIs, (p < 0.001). The readmission rate for TIs decreased during the study period. LPIs had a higher mean cost of birth hospitalization  and readmissions . For LPIs, birth hospitalization costs increased from 2007 to 2013, and decreased since 2014. Conversely, birth hospitalization costs of TIs steadily increased since 2005. The West region showed higher birth hospitalization costs for LPIs. Conclusions: LPIs continue to have a higher cost of birth hospitalization and readmission compared to TIs, but these costs have decreased since 2014. Standardization of birth hospitalization care for LPIs may reduce costs and improve quality of care and outcomes. In 2005, the National Institute of Child Health and Human Development recommended replacing the phrase “near term” with “late preterm ” to distinguish infants born between 34 weeks and 0/7 days and 36 weeks and 6/7 days from term infants (TIs) (≥37 weeks), acknowledging the vulnerability of the late preterm infants (LPIs) and helping redirect research efforts . The earHospital costs of birth are negatively correlated with gestational age . MoreoveThe objective of the present study was to examine previously unexplored trends of birth hospitalization and readmission costs for LPIs compared to TIs in the US between 2005 and 2016 using employer-sponsored private health insurance database. We hypothesized that we would observe a decrease in birth hospitalization and readmission costs, and readmission rates, for LPIs during this timeframe.® Commercial Claims and Encounters Database [The data in this study were extracted from the Truven Health Analytics MarketScanDatabase , which cInfants with birth hospitalizations were identified in calendar years 2005 to 2016 by extracting all inpatient claims that met predetermined inclusion criteria of admission type of “Maternity & Newborn”, age = 0, and relationship of child to the primary beneficiary. We used both International Classification of Diseases (ICD) diagnosis codes and DRG codes to identify that these claims were birth hospitalizations . Claims We excluded infants without ICD or DRG codes, or a diagnosis code indicating an unspecified week of gestation on birth hospitalization claim. Infants with a diagnosis code indicating <33 weeks gestational age or a DRG code of 790, indicating possible extreme immaturity, were also excluded from the study. Finally, we excluded infants with <30 days of continuous enrollment in MarketScan-covered insurance starting from the date of birth hospitalization discharge to study the trend of readmission costs of LPIs and TIs within 30 days of discharge.The clinical variables such as mode of delivery and multiple births were determined using ICD codes . On the We were primarily interested in studying the trend of healthcare cost of LPIs compared to TIs during birth hospitalization. Costs were measured from the perspective of the healthcare system and included total payments for claims eligible under the medical plan after applying rules such as discounts, including patient out-of-pocket payments. All costs from inpatient claims that occurred during the birth hospitalization were summed to create the total healthcare costs for an infant. All costs were adjusted to 2016 US dollars using the medical component of the consumer price index.For a small proportion of infants, the total healthcare costs were negative or 0 USD. Negative costs represent reimbursement or other adjustments to a previous error in billing. However, the error cannot be directly determined from the claims in which the reimbursement or adjustment occurred. These negative values are nonsensical for our purposes. To account for these values, we used Winsorization on 1.5% of the data, or 0.75% at each end of the distribution ,26. The Readmissions were defined as inpatient hospitalizations occurring in the first 30 days after discharge from the birth hospitalization. Costs during readmissions were also calculated in the same way as birth hospitalization costs (including Winsorization).For our primary goal of estimating birth hospitalization costs for LPIs vs. TIs, we used generalized linear regression models with a Gamma distribution and log link . The GamCost ratios (CRs) and 95% confidence intervals (CIs) were reported for LPIs compared to TIs from these models . While COur secondary goals were to examine readmissions rates and costs of readmissions. Readmissions were modeled using logistic regression with odds ratios (ORs) and corresponding 95% CIs reported . Costs oFinally, we also examined regional differences in birth hospitalization costs, readmissions, and costs of readmissions. Models were fit separately for each region in the database (Northeast (NE), North central (NC), South (S), and West (W)), with results shown graphically.n = 104,694 infants, 4.6%) and 33–36 weeks . After applying the continuous 30-day enrollment criterion, the final study cohort consisted of 2,123,143 infants with 145,201 (6.8%) classified as late preterm and 1,977,942 (93.2%) classified as term  were classified as term and 255,664 infants (11.2%) were classified as preterm with further sub-classification into <33 weeks  higher odds of readmission compared to TIs. We also examined readmissions for LPIs and TIs in calendar years 2005–2016 using a logistic regression model. Estimated probabilities from the model are shown in p = 0.002), indicating that the proportion of infants with a readmission differed across calendar year by cohorts. For TIs, readmission rates decreased significantly (p < 0.001) over the years. However, we did not observe a significant difference in trend of readmission rate across time for LPIs (p = 0.50). We observed a general trend of increasing estimated ORs of readmissions for LPIs compared to TIs from 1.77 in 2005 to 2.33 in 2016 . Based on the fitted regression model that included only gestational age, LPIs had 2.02-times . The LPIs had higher median (9300 vs. 6800 USD) and mean  readmission costs than TIs. Based on the fitted regression model that included only gestational age, estimated mean readmission costs for LPIs were 1.80-times greater than TIs (95% CI: 1.70 to 1.89), with corresponding mean cost estimates of 25,800  for LPIs and 14,300  for TIs.p = 0.75), indicating that mean cost estimates over time did not differ significantly by cohort. As shown in A regression model was fit to examine the readmission costs for LPIs and TIs in calendar years 2005–2016. Estimated CRs for readmission costs from the model are shown in Although the phrase “late preterm” was introduced in 2005, we continued to observe a trend of increased birth hospitalization costs for LPIs compared to TIs until 2013. Of note, we observed a recent decline in birth hospitalization costs of LPIs from the year 2014. In contrast, the birth hospitalization cost of TIs has steadily been increasing since 2005. Our study also demonstrates that LPIs were twice as likely to be readmitted compared to TIs. We observed a stable readmission rate for LPIs but a steady decline in readmission rate for TIs between 2005 and 2016. The readmission cost has steadily increased by approximately 3% for both LPIs and TIs since 2005. The West region showed a higher birth hospitalization costs for LPIs compared to other regions, whereas, no major geographical variation was noted in birth hospitalization costs for TIs and readmission costs for LPIs and TIs.Our study confirmed previous findings of higher birth hospitalization costs for LPIs compared to TIs regardless of study design, costing methodology, location and year of study ,29,30,31Our study also showed a 2x higher readmission rate for LPIs compared to TIs. For the current study, we focused on readmission rate and inpatient cost within 30 days of discharge from birth hospitalization for two reasons: (i) inpatient costs are the largest contributor (~92%) of the economic implications of preterm birth costs  and (ii)Our study has several limitations. First, our data include only commercially insured infants with employer-sponsored private healthcare plans; thus findings cannot be generalized to infants insured through Medicaid or State Children’s Health Insurance Programs. Of note, private insurance inpatient hospital payment rates follow the government insurance inpatient payment rates . TherefoIn summary, we continue to observe higher birth hospitalization costs, readmission rate and costs for LPIs compared to TIs. Moreover, there is a recent trend of decreasing birth hospitalization costs for LPIs, but the cost of readmission is still increasing. There is a consistent trend of decreasing readmission rate for TIs but birth hospitalization and readmission costs have increased over last decade. Given recent evidence of an increasing incidence of preterm birth, information on trends in the costs allow analysts and policymakers to plan clinical and budgetary services for the future. In addition, our data should be of interest to other researchers that are planning to evaluate the cost-effectiveness of new prevention and treatment strategies for LPIs.


Bunyavirales are negative-sense segmented RNA viruses infecting arthropods, protozoans, plants, and animals. This study examines the phylogenetic relationships of plant viruses within this order, many of which are recently classified species. Comprehensive phylogenetic analyses of the viral RNA dependent RNA polymerase (RdRp), precursor glycoprotein (preGP), the nucleocapsid (N) proteins point toward common progenitor viruses. The RdRp of Fimoviridae and Tospoviridae show a close evolutional relationship while the preGP of Fimoviridae and Phenuiviridae show a closed relationship. The N proteins of Fimoviridae were closer to the Phasmaviridae, the Tospoviridae were close to some Phenuiviridae members and the Peribunyaviridae. The plant viral movement proteins of species within the Tospoviridae and Phenuiviridae were more closely related to each other than to members of the Fimoviridae. Interestingly, distal ends of 3′ and 5′ untranslated regions of species within the Fimoviridae shared similarity to arthropod and vertebrate infecting members of the Cruliviridae and Peribunyaviridae compared to other plant virus families. Co-phylogeny analysis of the plant infecting viruses indicates that duplication and host switching were more common than co-divergence with a host species. Bunyavirales infect arthropods, plants, protozoans, and vertebrates. Their RNA genomes are segmented and exhibit negative or ambisense polarity. Each virus species has a fixed number of genome segments which range from two to eight, with plant viruses having the largest numbers of segments. The nucleotide sequences at the 3′ and 5′ terminus of each genome segment are complementary and form panhandle structures for stability. Their RNA segments are mostly coated in nucleocapsid proteins and further encapsulated in an envelope derived from its host cell.Viruses in the order Bunyavirales is a recently established taxonomic order that encompasses twelve families comprising 46 genera [Hantaviridae, Nairoviridae, Peribunyaviridae and Phenuiviridae [Bunyamwera virus (BUNV), Crimean-Congo haemorrhagic fever virus (CCHFV), Hantaan virus (HTNV), La Crosse virus (LACV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome virus (SFTSV), and Sin Nombre virus (SNV). Three families within Bunyavirales contain members that infect plants as their primary host: Fimoviridae, Phenuiviridae, and Tospoviridae. Within these families, there is one genus of plant infecting viruses: Emaravirus, Tenuivirus, and Orthotospovirus, respectively. Across Bunyavirales, viruses can have three segments of negative-sense or ambisense RNA that are named according to their relative length. These segments are known as large (L), medium (M), and small (S) which encode the viral RNA dependent RNA polymerase (RdRp), a polyprotein precursor glycoprotein (preGP) that is co-translationally cleaved into two mature glycoproteins (Gn and Gc), and the nucleocapsid (N) protein, respectively. Among the plant viruses with more than three genome segments where “x” equals the total number of segments, each segment is numbered from RNA1 to RNAx and are assigned sequentially to each segment in order of decreasing nucleotide length. A novel genus that is tentatively named Coguvirus has a bi-partite genome, lacks an external envelope and, is proposed to the order Bunyavirales [6 genera ,2. Four iviridae ,2. Theseavirales ,4.Bunyavirales are unclear. While phylogenetic studies suggest common ancestral origins of vertebrate and arthropod infecting viruses, the ancestral lineages of plant viruses within this taxonomic order have not been well studied [Bunyavirales [The origins and evolutionary history of plant viruses within  studied ,6. Exten studied ,7,8,9,10 studied . While tavirales ,12,13,14Bunyavirales, which has contributed to the recent reorganization of families within this taxonomic order [Bunyavirales by examining the shared and conserved hallmark genes among arthropod, plant, protozoan, and animal-infecting counterparts. This study also includes analysis of plant viral movement proteins which represent important changes in virus evolution from deeply rooted ancestral viruses.Recent research in the field of virus metagenomics has expanded the list of new plant-infecting species within ic order . This stBunyavirales by the International Committee on Taxonomy of Viruses (ICTV) [We retrieved RdRp, preGP, N, and MP sequences from the NCBI protein archive . We useds (ICTV)  as a guis (ICTV) ,16,17 uss (ICTV) ,19. Prots (ICTV) . Phyloges (ICTV) ,22. Trees (ICTV) ,24. ImagThe 3′ and 5′ UTR regions of viral segments were extracted using NCBI nucleotide database . SequencFor calculating identity scores of MP amino acid sequences, pairwise sequence alignment was performed using the software Sequence Demarcation Tool (SDT) v. 1.2 .Cophylogenetic relationships between families and their natural hosts were analyzed with event-based co-phylogeny analysis tool Jane ver. 4.01 . PhylogeBunyavirales, RNA1, (or the L segment) is the longest and encodes RdRp. The RdRp sequences for 253 species belonging to arthropod, plant, protozoan, and vertebrate infecting viruses within Bunyavirales were compiled (Chilibre phlebovirus (CHIV) is classified by the ICTV as a member of the family Phenuiviridae but the ML tree indicates that the RdRp is in the lineage group I with Peribunyaviridae family and clusters with the Pacuvirus and Herbevirus genera. This unusual relationship, verified using the aLRT-SH test  [Myriapod hubavirus, Haartman hartmanvirus, and Striated antennavirus are at the deepest root supporting lineage group IV viruses. The invertebrate-infecting species Millipede wumivirus is at the deepest root supporting lineage group V.The next deeply rooted clade includes groups IV and V, . (2017) . The invLeptomonas shilevirus and Laurel Lake virus at the deepest node. The plant-infecting genera Tenuivirus and Coguvirus and the insect-infecting genus Goukuvirus are the closest relatives to these invertebrate-infecting genera [Phlebovirus genus represents a smaller fraction of viruses within this lineage group with the majority of viruses representing plant, insect, and protist-infecting viruses [The third major branch has the invertebrate-infecting species g genera . While G viruses . The RdR viruses .Fimoviridae and Tospoviridae, we carefully examined an alignment of their RdRp sequences. Crystal structures of several members of the order Bunyavirales have been used to identify functional motifs and similarities across species within the order, and these reports informed this analysis [Fimoviridae and Tospoviridae share the conserved motifs (H…PD…D/E…K… T/Y…Y) in the endonuclease active center occurring in all families of Bunyavirales [Orthobunyavirus, Orthotospovirus, Hantavirus, and Phlebovirus genera have the conserved D/E motif between the H and PD  encodes the preGP which is inserted into the host endoplasmic reticulum (ER) and cleaved by the cellular signalase into Gn and Gc [Nairoviridae and Peribunyaviridae, the NSm is part of the polyprotein located between the Gn and Gc regions. For Phasmaviridae, NSm is located at the N-terminus of the Gn sequence. For Tospoviridae, the NSm is ambisense oriented and located next to the Gc domain. For Phenuiviridae, the NSm is an open reading frame nested within the Gn region. Members of the genera Tenuivirus and Emaravirus lack the NSm coding sequence in RNA2 and M segment  are not available. The Gouleako goukuvirus, which is the type member of the genus Goukuvirus (family Phenuiviridae), is a deeply rooted branch that precedes the major subclades in groups I, II, and III. Looking at the M segment (or RNA2 segment) for each genus within these virus families, the length of the preGP varies significantly. In group I, Orthonairovirus fall into two classes that either contain or lack the NSm within the polyprotein  and Phenuiviridae. From the Pacuvirus branch, there are three major subclades: three species of Orthobunyavirus cluster in group VI, the group V Phenuiviridae cluster, and the group IV cluster of primarily Hantaviridae with sole representatives of Arenaviridae and Mypoviridae  across all families ((14)-D-X(52–63)-W, while the tenuiviruses have a submotif Φ-X-Φ-P-D. The W residue is not conserved downstream of the D motif in the tenuivirus MPs  had identical terminal sequences. It is interesting to see such conservation among animal, plant, and arthropod-infecting viruses. The species CHIV within the Phenuiviridae, which we repeatedly noted to be misclassified phylogenetically with Peribunyaviridae, also shares the identical terminal sequences with these virus families. Additionally, the plant-infecting Phenuiviridae (Tenuivirus genus) and Coguvirus share identical 5′ ACACAA/G and 3′ U/AUGUGU terminal sequences.The coding regions of each genome segment lie between terminal non-translated sequences that vary in length. The 3′ and 5′ genomic RNA termini are essential for RNA synthesis and are typically invariant. We compiled the terminal 20 nucleotides for all species that were used in the phylogeny into a table, leaving gaps for those whose sequences were not reported . We thenTospoviridae, Arenaviridae, and Myopviridae are unique . It is also worth noting that there is only one nucleotide difference between the 5′ and 3′ terminal sequences of Nairoviridae and Wupedeviridae. The Nairoviridae has 5′ UCUCAA and 3′ UUGAGA while Wupedeviridae has UCUCUA and UAGAGA. The terminal nucleotides for e unique . NotablyArenaviridae, Fimoviridae, Hantaviridae, and Phasmaviridae than co-speciation . Duplication is more common than co-speciation or host switching for Arenaviridae, Fimoviridae, Nairoviridae, Peribunyaviridae, Phenuiviridae and Tospoviridae. Considering the preGP, N protein, and MP phylogenies show that vertebrate and plant infecting viruses are related to arthropod infecting viruses suggesting that cross-species transmission may occur between arthropod species, plant species or vertebrate species. However, there is little evidence to suggest the cross-kingdom movement of viruses. The tree also revealed between plant and vertebrate hosts but clustering, host switching during evolutionary history could support the divergent phylogenetic positions for some species within the taxonomic families. Surprisingly, the analysis suggests extinction plays a major role in the evolutionary history for all families in Bunyavirales except for Phasmaviridae , duplication events (green), host switch events (blue) and loss events (purple) were summed for each family. Boxes represent the estimated median (center line) interquartile range (IQR) and whiskers represent 1.5× IQR.A. tree of viral segments harboring hallmark genes and movement protein.B. Maximum-likelihood Bunyavirales. The genome segments were concatenated in silico before analysis. The virus’ vector is listed to the left of the tree, and species with an asterisk (*) have an unknown vector. Each virus is color-code based on its plant host type: monocots (red), superrosids (yellow), or superasterids (blue).A maximum-likelihood phylogenetic tree was constructed from the complete viral genome segments that encode hallmark genes and movement protein (if the sequence is available) for each plant-infecting virus within the order Bunyavirales. We focused on the genome segments L (or RNA1), M (or RNA2), and S (or RNA3) encoding the RdRp, preGP, and N proteins respectively because they consistently define all members of Bunyavirales. We included the analysis of the viral MP because they are a defining feature of plant infecting viruses. This research is timely because, in 2019, the order Bunyavirales was amended with significant changes in the associated numbers of families, genera, and species [This study examines the phylogenetic placement of plant viruses within the order  M or RNA, and S  and the Chilibre phlebovirus share identical terminal 6 nucleotides and the RdRps reside in Groups I, II, and III which derive from a common deep-rooted branch. The Nairoviridae and Wupedeviridae in Group V RdRp have identical termini except for one nucleotide and the RdRp Group VI Phenuiviridae and Coguvirus share identical termini. The RdRp Group II and III affiliated Tospoviridae, Hantaviridae, and Phasmaviridae have unique terminal sequences that are shared within these taxonomic families. It is also interesting to point out that the 3′ and 5′ terminal sequences of the plant infecting Fimoviridae share identity with the vertebrate infecting Peribunyaviridae and not the plant infecting Tospoviridae. This observation suggests that the high degree of sequence identity within the RdRp endonuclease and polymerase catalytic motifs of the Fimoviridae and Tospoviridae is not the driving force for co-evolution of the terminal UTR sequences [Fimoviridae, Tospoviridae, Phenuiviridae, and the unassigned Coguvirus, experiments are needed to understand how the functional roles of the terminal UTRs.The RdRp and N form a highly stable complex with viral RNAs that are packaged into virions . The iniequences . Howeverequences . For thePhlebovirus; family Phenuiviridae. Members of the genus Phlebovirus are viruses that are borne by ticks, mosquitoes, and sandflies. Prior molecular characterization suggested that CHIV may be more related to the genus Pacuvirus within Peribunyaviridae [Pacuvirus within the Group I Peribunyaviridae. The preGP also shows a strong phylogenetic relationship with Pacuvirus and Orthobunyavirus in group VI. This ML analysis supports the suggestion that the taxonomic identity for CHIV should be moved from the Phlebovirus to the Pacuvirus genus within Peribunyaviridae [Chilibre virus because these viruses share common reservoirs [The CHIV is assigned to the genus aviridae . Our ML aviridae . Inter-lservoirs . Howeverservoirs ,57,58.Phenuiviridae, the preGP resides in four groups while the N proteins reside in six different groups. Focusing on the plant infecting viruses, the Tenuivirus N proteins extend from a deep branch which at its base bifurcates to the insect and vertebrate infecting Webuvirus, Pidchovirus, and Orthohantavirus [Emaravirus and Orthophasmavirus preGP and N proteins suggest a similar ancestry. The Orthotospovirus preGP proteins are phylogenetically positioned near the Orthonairoviruses and Mammarenaviruses while the N proteins extend from a node that is affiliated with two dipteran infecting virus members of Phenuiviridae in group II [Bunyavirales involve a network of gene exchanges. Such gene exchanges likely led to the emergence of new virus species. The data in Arenaviridae and Phenuiviridae within several phylogenetic groups and strongly suggests the exchange of genes between viruses. An evolutionary mechanism of recombination is supported by the presence or absence of NSm either fused or nested within the preGP coding sequences of neighboring virus genera within a phylogenetic group.Interestingly, the preGP and N protein phylogenies each exhibited a higher extent of diversity with members of the same taxonomic family sometimes represented in more than one lineage group. For ntavirus . The phygroup II . These oRice grassy stunt tenuivirus and orthotospoviruses are closely related in the ML tree. The pairwise analysis also shows that the MP similarities cluster mainly according to the virus genus. All of the MPs in this study have been ascribed to the 30K superfamily of viral MPs which share a common aspartic acid residue that is commonly known as the D motif [(15)-D-X(53–59)-W, while the tenuiviruses have a submotif Φ-X-Φ-P-D. Analysis of the plant viral MPs shows three lineage groups and surprisingly the MPs of  D motif ,48. We iBunyavirales might occur by recombination or reassortment of segments [Fimoviridae shows duplication, and host switching occurs more than co-divergence while Tospoviridae and Phenuiviridae show very little host switching. Extinction was high for most families in Bunyavirales and this outcome can occur if there is an incongruency between the virus and host phylogenies, when invertebrate vectors narrow the niche diversity, or spill-over infection leads to a dead-end [Until now horizontal gene transfer among positive-strand RNA viruses and double-strand RNA viruses has been well described but there has been little evidence of gene exchanges among negative-strand RNA viruses ,60,61. Hsegments ,57,62. Mdead-end ,59,63,64dead-end . The tendead-end ,49. Orthdead-end ,66,67. TBunyavirales. The phylogenies reveal extensive conservation among the hallmark genes of plant-infecting viruses with insect and vertebrate counterparts. The phylogenies reveal important insights into the strength of virus–host and virus–vector interactions. Further research is needed to understand the potential for horizontal gene transfer across diverse virus lineages. A priority for future research is to understand the barriers to virus and host co-speciation that could be critical for preventing epidemic virus spread.Our findings provide a comprehensive view of plant virus phylogenetic relationships within the higher ranking of the order


The head louse infestation is a public health issue in the world especially, affecting most people who live in camps, school-aged children and their families. Head lice treatment has economic ramifications that often under calculated. The aim of this study was evaluation of economic burden associated with head louse infestation in Iran.In a cross-sectional study, 500,002 infestations were diagnosed among suspected head lice infested people who referred to health care system in all provinces of Iran during 2017. Direct and indirect costs related to paid by patients and government systems were extracted by referring to accounting documents and interviews with patients and experts and were recorded in researcher-made forms. Microsoft Excel 2010 software was used for economic burden calculation.The incidence rate of head lice infestation in Iran was 500,002/79,926,270 . Economic burden of head lice in the country was calculated at 5,790,143$. Direct and indirect costs, governmental cost, out of pocket and total costs of head lice were included 3.14$, 2.84$, 5.98$, 5.60$ and 11.58$ per case respectively.The direct and indirect costs associated with treatment of infestations were relatively high. Therefore, the creation of medical facilities such as availability of diagnostic and treatment strategies can be effective in the control of infestation. The adoption of infestation prevention methods, such as health education to people at risk of infestation, reduces the incidence of head lice and imposition of related treatment costs on governmental health care system and head lice cases. Pediculus humanus capitis (Phthiraptera: Pediculidae) is an obligate ectoparasite of placental mammals especially humankind. This blood-feeding insect does not survive away from the body’s host or loses its infestation , Argentina, Brazil, the United Kingdom, Australia and Turkey . Many stThe calculation of the economic cost associated with disease is used to determine the burden of disease in societies. Totally, economic costs of care associated with head lice infestation are included direct and indirect costs. Direct costs due to diagnosis and treatment of disease and indirect costs related to lost time from work and school . In addiThis study aimed to estimate the economic burden of head lice in all provinces of Iran.2. Based on the most recent census in 2016, the country has a population of approximately 79,926,270 out of which 68.4% inhabit in urban areas and 31.6% in rural environs  after two applications almost a week apart was considered as treatment success . The cosAfter holding several meetings with the technical deputy of the CDC, experts in the prevention & control diseases, network expansion plan and pharmacy departments, data has been obtained and recorded in the researcher-made forms. Then the financial records were returned to the finance department of CDC. The collected data were entered into Microsoft Excel 2010 software. Ultimately, direct and indirect costs associated with health care to patients were calculated. Direct costs due to diagnosis and treatment of infected people were included expenses related to out of pocket , personal costs, providing head lice treatment products such as permethrin shampoo as a first-line choice for treating head lice infestation. Indirect costs related to lost time from work and school. In addition, lost wages for parents, costs of day-care for parents who have to employ nurses at home because they cannot miss work are included as indirect costs. Expenses related to consumable materials and supplies (note books and stationery), public utility costs  should be added to indirect costs. In addition, overhead costs include building maintenance costs , transportation costs and depreciation costs of buildings and equipment. Microsoft Excel 2010 software was used for the costs calculation.Totally, 500,002/79,926,270  infested head lice patients were diagnosed and recorded among suspected cases to human head lice infestation who referred to health care system in all province of Iran during 2017.The infestations were higher in six provinces including Kerman, Hormozgan, Sistan & Baluchestan, Khuzestan, Bushehr, and Qom, mainly located in southeast, south and central Iran , Fig. 1.The economic burden of all infestations was estimated at 289,507,158,024 rials  over a one-year period in the country. On average, direct costs of providing health care services to all head lice infested cases were calculated for each person (Expenditure per capita) was 157,337 (3.14$).Indirect costs (overhead) per person were about 141,675 rials (2.84$), the governmental cost (direct & indirect costs) per patient was 299,012 rials (5.98$), out of pocket was about 280, 000 rials (5.6$) for each patient, and the average total cost during this period was estimated at 579,012 (11.58$) rials per case.Direct and indirect costs related to this public health problem were close to each other, so that 39,630$ (52.6%) of the costs were direct costs and 35,685$ (47.4%) also included the cost of overhead treatment for head lice infestation . The ratIn present study, prevalence of head lice infestation calculated at 625.5 per 100,000 populations (0.62%), which was relatively lower than the incidence rate in the Asian countries (varied from 0.7% to 59%) . In IranOne of the limitations of this study was the failure to calculate cost of patients’ lost time. Lost school days and lost their work days that head lice infested people must stay home to rest and care for children came back home from schools because of their infestations should be added. In addition, other intangible costs were costs of mental disorders due to this health problem were not considered in the study that is another limitation of the study.Direct and indirect costs associated with the diagnosis and treatments of head lice infestation are high in Iran. Therefore, considering prevention and control strategies such as health education to people at risk of this health issue about types of human head lice transmission and early detection of this infestation, can reduce the costs on patients and health care system. In general, this plan is more affordable. In the head lice infestation program, facilities such as the free-of-charge diagnosis and health planners and policy makers will target treatment strategies to reduce out of pocket.Ethical issues  have been completely observed by the authors.


Recent seroprevalence studies in animals detected Rocio virus in regions of Brazil, indicating risk for re-emergence of this pathogen. We identified Rocio virus RNA in samples from 2 human patients for whom dengue fever was clinically suspected but ruled out by laboratory findings. Testing for infrequent flavivirus infections should expedite diagnoses. Flavivirus, such as dengue (DENV), Zika, and yellow fever viruses, along with co-infections with other arboviruses (Brazil has been affected by outbreaks caused by viruses of the genus Rocio virus (ROCV) is a potentially emerging neurotropic flavivirus in Brazil; however, because relatively little is known about the biology of this virus, technologies for its detection are limited , and the male patient experienced headache, eye pain, pruritus, nausea, and leukopenia   . Both paThe molecular diagnostic result for positive ROCV in humans reported in this study corroborates the results of other studies involving serologic tests for ROCV in animals (Supplemental methods and results for study of detection of Rocio virus SPH 34675 during dengue epidemics, Brazil, 2011–2013.


To analyze the initial healthcare response to the Zika virus in Piura, Peru, and assess the perceptions of midwives and nurses regarding their role in prevention of Zika virus and management of congenital Zika syndrome (CZS).This ethnographic study used a rapid qualitative assessment design. Data were collected through a focus group with midwives and in‐depth interviews with midwives (n=11) and nurses (n=5).The focus of the early Zika virus response in Piura was on pregnant women and vector control. Midwives received some training on Zika‐related care during the early response. Nurses did not receive any Zika‐specific training. Neither nurses nor midwives were trained in neonatal CZS surveillance. Midwives were clear about the value and feasibility of incorporating Zika virus surveillance in their daily work, however nurses were not. They referred to lack of training and appropriate tools as limitations. Confusion about Zika virus and CZS symptomatology and effects persisted in both groups. Concerns about their own personal risk influenced the ways they engaged with Zika virus prevention in the community.Long‐term management of endemic Zika virus in Piura will require the engagement of both nurses and midwives as primary care providers. A cost‐effective and culturally competent approach should include a broader focus on family planning and child development surveillance. This requires cross‐disciplinary collaboration between nurses and midwives. Zika virus infection during pregnancy has been linked to stillbirths and to congenital Zika syndrome (CZS) characterized by varied levels of cognitive impairment, microcephaly, and a host of vision, hearing, and motor impairments. Additionally, Zika virus infection in nonpregnant women has been linked to an increased incidence of Guillain‐Barré syndrome.Health providers are the first line of care and surveillance for the long‐term management of any endemic disease, yet little is known about how they process and view their roles in cases of emergent and complex diseases. Zika virus is one such disease. Primarily transmitted through the bite of an infected The characteristics of Zika virus infection present new challenges for the public health response and long‐term prevention. A large number of asymptomatic cases,Between 2016 and 2017 around 6000 cases of Zika virus were reported in Peru, with higher concentrations in the regions of Loreto, Piura, Ica, and Cajamarca.In Piura, there were around 300 confirmed cases of Zika virus infection between 2017 and 2018, and 88 so far in 2019.The aim of the present study was to analyze the initial healthcare response to the Zika virus epidemic in Piura and assess the perceptions of frontline personnel—midwives and nurses—regarding their role in prevention of Zika virus and management of congenital Zika syndrome (CZS).2The data reported here were part of a larger study that included focus groups and interviews with local men and women, and health providers. The present article reports only health provider data collected in two phases: phase 1, August–September 2017; and phase 2, January–February 2018.This ethnographic study used a rapid qualitative inquiry design, featuring intense but shorter data collection than traditional anthropological ethnography.The location of the study, the Catacaos Micro‐Network, is part of the Lower Piura Health Network. The micro‐network is comprised of an urban clinic that includes all primary care, pregnancy, birth care, and surgery facilities, and six dependent health centers, with lower levels of care, located in outlying and/or rural areas. Names of participants and their health centers have been omitted in accordance with Institutional Review Board recommendations.A summary flowchart of the study is given in Figure The focus group questionnaire was generated to understand the context of Zika virus interventions and served to create an in‐depth interview guide. The focus group was conducted in a meeting room at the Catacaos clinic and lasted approximately 90 minutes. Interviews asked about initial Zika response, knowledge of Zika, perceptions about clinic‐based Zika virus prevention, and perceptions of how Zika virus has affected the midwives’ and nurses’ personal and professional lives. Interviews took place where participants felt comfortable—most in their workplace after hours and some in a nearby cafeteria. Each interview took 45–70 minutes. All participants provided verbal consent and received a study information sheet in Spanish. Field notes were taken (RI) during the interviews to register main ideas and contextual information. No other people were present during data collection. Recruitment for interviews continued until saturation was reached.Data were recorded, de‐identified, and transcribed verbatim. Transcriptions were coded deductively for predetermined and emergent codes using ATLAS‐ti version 8 . Focus group data served to understand the context and create a semistructured interview guide. All data presented here are from in‐depth interviews. The authors coded independently, and coding structure was discussed and harmonized at two points during the process. Codes were organized into categories in response to research questions. Major themes in each category were derived from the data and represent shared experiences Fig. . TranscrThe study was approved by the Pan‐American Health Organization Ethics Review Board and the Ethics Committee of Peruana Cayetano Heredia University in Lima, Peru. The protocols and informed consent forms were shared with regional partners and assessed for cultural concordance by local research team members.3All midwives and nurses in the study were female, with an average age of 42 years. The average age of the midwives was 47 years, whereas for nurses it was 34 years. Of the 17 midwives and 5 nurses, 14 were partnered and 14 had children. Most had worked in the Catacaos health micro‐network for between 1 and 25 years.Midwives and nurses have clearly delimited professional responsibilities in urban, well‐staffed facilities, like the Catacaos clinic. Midwives oversee sexual health, family planning, and prenatal and obstetric care. Nurses handle neonatal and primary pediatric care. However, at rural facilities, often one nurse or midwife was in‐charge of providing all care to women and children.3.1Midwife 11The patient comes to her first control; everything is explained to her, we give her advice […] she has to dress up, at night even though is hot, avoid being bitten by the mosquito, put on her mosquito net, use the repellent, have sex with the husband with a condom. The increased risk posed by Zika during pregnancy meant that the initial response involved midwives directly, but not nurses. During early Zika virus interventions (May 2016 to May 2017) most midwives interviewed received training in Zika virus symptoms and counseling, and received mosquito nets, bug repellent, and condoms for their pregnant patients. Local flooding followed by an outbreak of dengue in 2017 disrupted Zika virus surveillance and repurposed all personnel to the dengue response. However, features of this early training were incorporated into midwives’ ongoing pregnancy care protocols. As M11 stated, midwives were trained to provide this information in the first prenatal visit:Nurse 4The girl, the girl's growth, the head circumference was not improving. She was only one month old […] The [doctor] saw her and they sent her to the lab and there I think they confirmed something, or maybe not and then the mother was sent to gynecology. From there I don't know, I lost track and did not follow up. Nurses did not participate in early Zika virus training. Interviewees anecdotally described how they learned about Zika virus through the media and colleagues. They assumed that they would likely be involved only if any cases of microcephaly occurred. However, even in cases of suspected microcephaly, care was derived to specialists, and primary care personnel lost track of cases:3.2Aedes aegypti mosquito, like dengue. However, it was frequently confused with dengue. For example, M6 reported:Midwife 6Zika is another type of dengue, more aggressive and what's worse it attacks women in the first trimester and produces microcephaly. Interviewees knew that Zika virus is transmitted by the Midwife 7[…] fever, increase temperature, joint pain. But only the lab can tell us the difference with dengue […] because it is the same symptoms, almost. Though maybe that [gestures towards arm] rash, although you get that with dengue too, so only the lab will tell you. Similarly, M7 said that the symptoms are:Midwife 8Well, Zika is a disease transmitted by the same mosquito that transmits dengue, yellow fever and chikungunya. In a pregnant woman it is very dangerous because it directly affects the baby. What the virus does to the baby, it eats the brain. That's what I say to them. It destroys the brain, and the child can be born without the ability to see, it doesn't hear, doesn't talk, doesn't walk. And it also depends on the gestational age when it is affected. That it is very important that they take care to not get bitten by a mosquito, because we are in an endemic area, because we had had many cases of dengue and because that is the same mosquito that transmits Zika, then we are not free [of risk]. The other thing that is very worrisome is that it is a virus that doesn't produce many symptoms, maybe women will feel a slightly higher temperature that can be dismissed, but that something that can indicate [Zika] is the appearance of skin rashes. That is what we tell them. Some midwives, those working in the urban clinic, had received more training and could identify skin rashes as a key symptom of potential cases to differentiate Zika virus from dengue:However, no midwife indicated that rash on the palms is one of the best ways to distinguish symptomatic Zika virus infection. It is encouraging that almost all midwives did identify the sexual transmission of Zika virus.Nurse 4I know that Zika is viral, the same vector as dengue and chikungunya. All the Americas are endemic. Pregnant women are more vulnerable and the child is born with microcephaly, so the damage when infected is really severe. [Where did you get that information?] From the internet mainly. I like to know how new things will affect us. Nurses had little knowledge about Zika virus; they only identified mosquito‐borne transmission of the virus and did not mention any other forms of transmission. They had not received any specific training on Zika virus, yet some had found information from other sources. For example:Nurse 2As far as I know, no Zika has arrived here, [mainly] because it's the doctor who monitors things [like] high fever I haven't touched any Zika cases so far, none, or dengue either. [Symptoms of Zika are] bone pain, nausea, vomiting I think, I don't remember the symptoms of Zika very well, just something, not very well, I would be lying. Nurses knew very little or nothing about CZS, with the exception of the presence of microcephaly. There had been few cases of Zika virus, and these had been managed by medical doctors, which may have impacted the ability of nurses to identify symptoms, for example:Nurses’ lack of knowledge of Zika virus and CZS symptomatology is concerning given that they are often the only primary care professional in lower‐level health posts in rural areas, and because they are in charge of caring for neonates and infants. Interestingly, neither nurses nor midwives discussed Guillain‐Barré syndrome as an effect of Zika virus.3.3Nurse 1So, then we work according to the ‘febrile patient’ protocol […] In triage we capture the symptomatic patient or the febrile ones. If a febrile patient presents in triage the technician will immediately ask “Where were you in the past few days?” And according to that, if there are any other associated symptoms, like joint pain, red eyes—that would be conjunctivitis—we then fill out an epidemiological form according to the dengue protocol. We are more careful if the patient is a female in reproductive age and even more if she is pregnant […] because we know that Zika is transmitted through sexual intercourse and [we take a lot of care of] the baby. Because of congenital malformations. Then according to that personnel derive the patient to the physician. Primary care personnel are necessary for vector‐borne disease surveillance including Zika virus. Given Piura's long experience with dengue and malaria, there is an acute awareness of the importance of identifying febrile cases. Currently the same strict protocol is used to identify potential Zika virus infection cases, despite the high level of asymptomatic cases and the lower likelihood of high fever. This protocol relies heavily on medical doctors for diagnosis and referral:Nurse 1The issue is that this is a level 2 [low level rural] clinic, we have a SERUMS doctor they see patients only in the morning or the afternoon. So then the nurse [me] has to step in. Rural health centers generally do not have a permanent medical doctor and refer patients to the main clinic or “head of the network” for basic triage and diagnosis. At least three interviewees noted that the physician in their rural clinic was a SERUMS—a recent medical graduate completing their rural and peri‐urban practice year. These doctors have limited experience, work limited hours and for only one year, thus they are not fully part of the rural healthcare team. For example:Midwife 10Right now, I have a case of a patient, exact same as another, with malformations, there's a sonogram that shows malformations, I'm just about to notify epidemiology. But I don't have any confirmation. I only have those two cases, but in sonograms there are malformations that lead us to assume [that maybe there's more]. We would need to do a study to confirm. Here we notify epidemiology and nursing, and then epidemiology takes over coordinating with the Santa Rosa Hospital. […] They should return with a counter‐referral form. But, I'll be honest, that never happens. They receive care over there [hospital] and we maybe do a little follow‐up. Often patients don't tell you anything because it's all medical terminology, [they just say] “they told me it [baby] was fine.” Once referral procedures take place, patients rarely return to the primary care facility with a clear diagnosis or instructions to allow follow‐up from nurses and midwives. Many patients don't remember or understand what the outcome of the referral was, and the primary care personnel do not receive any counter referral indications:In this case and that of Nurse 1, health personnel expressed exasperation and confusion as to their current role in Zika virus care. The lack of an efficient and comprehensive referral and counter‐referral system is perceived as a barrier to effectively engage in Zika‐related care.3.4Midwife 6[we are concerned] as you see, here is our repellent, every time we come here [clinic] the first thing we do is zzzzzzz [apply it], the first thing we do. It is the most common measure that we all do here, not only me, we all have our repellent. As for food, we have also told everyone to drink acidic juices. Here [I have] orange, lemon, we try to drink [these] citrics [to remain healthy]. One key issue is the relationship of health personnel with the community and their own risk awareness. Most midwives and nurses live near where they work, they are exposed to the same risks and share similar cultural perspectives as their patients. Many are concerned about their own well‐being and care. For example, discussing their own constant vigilance for mosquito bites:Midwife 11Yes indeed, because we do outreach work. With the flood and everything, we did not have the Health Center, it did not work because it was flooded, and everything was spoiled. So, we have to go out to the campaign, go out to the shanty towns that belong to a rural community and there were a lot of mosquitoes, with the water and everything. And yes, we were very worried; at least we protected ourselves, we came with a long sweater, we used our repellent but still there is always the worry and we got it, we got dengue no matter how much we used the repellent, no matter how much we used the mosquito net. We got dengue anyway because the mosquito is there and there were plenty mosquitoes, there were a lot of Aedes. However, most of these precautions center around concern for dengue. During the floods of 2017, almost a third of interviewees and many of their family members became ill with dengue. They attributed this contagion to outreach work in flooded rural communities:Midwife 7I can't have any more children even if I wanted to [sterilized] but it does worry me for my children who are young, thirteen and fifteen. Midwife 8I am 48 my husband is 52 I am not concerned for myself; we are not getting pregnant again. But I have two nieces who were pregnant. I was concerned for them, they live in [rural areas] and they had many mosquitoes, but thankfully everything turned out ok. Concern over the Zika virus contagion was varied among nurses and midwives. Those who were past reproductive age or were sterilized were not concerned for themselves, but for their daughters or other family members:it is a disease that can lead to death.” Nurse 3Others conflating dengue and Zika were concerned about it because they believed that: “Midwife 5Well it hasn't meant many changes in my life really, because we are already taking preventative measures for dengue. The only thing is that I'm not pregnant, my husband uses condoms. […] we use rhythm method and condoms only on fertile days. Overall, those that were still of reproductive age echoed patient attitudes and did not take any extra precautions to avoid pregnancy:Midwife 2I would be worried if I was pregnant. I am more concerned about dengue. When we go to community outreach, we use insect repellent. […] I use the monthly shot and sometimes condoms [so Zika is not a concern]. Some reported that already having a secure contraceptive method, like the implant or the shot, gave them a sense of calm since they were already avoiding pregnancy:Overall, perceptions of risk among participants demonstrated little concern for Zika virus infection, though general concern for mosquito‐borne diseases, and dengue specifically, was very high.3.5Midwife 7Here we focus on pregnant women. […] But us, as midwives, we focus on our pregnant women. We prevent the same as dengue, we talk about how they could contract dengue from the mosquito bite, but also through sexual intercourse. We gave them, talked about prevention with them regarding their protection, first with insect repellent, with mosquito nets, closing doors, avoiding standing water. Midwives had a clear perception of their responsibility in Zika virus prevention since it is part of their protocols for prenatal counseling. However, they mainly see Zika virus as related to the care of pregnancy and not necessarily to family planning or reproductive counseling:This midwife echoed the need to protect pregnancies, and interestingly also conflates dengue and Zika.Midwife 6Yes, [we have done] outreach to schools, for teenagers and then children, mothers clubs [the message is] to get rid of mosquitos, get rid of Zika. Midwives recognize the need to extend prevention messages to nonpregnant women. However, many of these initiatives were bundled into the educational outreach related to the dengue outbreak, and they focused mostly on pregnancy care, and vector control:Midwife 4[the main messages] are for our pregnant patients definitely, and it's malformations. We want healthy babies, so they have to use all the ways to prevent it and take care. [we tell them] how they must keep water reservoirs, to use mosquito nets, bug repellent, and condoms. While condom use was mentioned, it is clear that midwives mostly considered it only as part of the messages for pregnant women, not part of a general pregnancy delay strategy:While contraceptive counseling and promotion of family planning methods are a central part of the midwives’ work in the community, none mentioned them as a part of Zika virus prevention.Nurse 5The truth, since you asked, we have not done anything. The truth, it would be good to do something, we have not thought about it. We have seen it as something that is real, of course, but not enough to give information or give informative talks to our mothers. We had not contemplated it, to be honest. But right now, we talk about it like that, we break it down, and you're right. We could start with one more topic, such as breastfeeding, child's care, caring for this [Zika virus]. Nurses, however, did not directly consider Zika virus prevention as a direct part of their responsibility, a perception reinforced by their lack of training:Nurse 3Well, we haven't actually seen any Zika in children, mostly in pregnant women. But we do recommend hand washing in all office visits. Hand washing and changing water in their reservoirs at home [so it's not standing for long] and we tell them about the symptoms and such, and to go to emergency care at the health center. Those that had did make efforts to discuss Zika virus felt most comfortable talking about preventing mosquito bites and managing vector control:Nurses see more of a role for themselves in CZS surveillance than in working with pregnant women.3.6Midwife 11So, we would go out to the streets looking for pregnant women, those in their early pregnancy, which is when the effect [of Zika virus] on the product is greater. We would give them counseling, and we would also give them condoms; because of course sex is still happening, but you have to avoid it, the infection. Many of the husbands of the women work outside the community, maybe in Tumbes, or on the border, and there were foci [of Zika virus infection] in those areas. So, to avoid that after counseling we gave them condoms. We haven't had any cases so far here in this area [small rural hamlet], but we have worked really hard. Both nurses and midwives considered that community work should be a part of future Zika virus prevention. They are already engaged in what they call “extramural” work, especially in rural and peri‐urban areas where access to health care is limited:Midwife 11So that's how it was, we worked with pregnant women, with the general population. We do the “ten‐minute intervention.” [Health professionals] go to each house for ten minutes, to check for discarded materials, check for mosquito breeding areas. They [the community] save water because here we don't have water all day. So, there are checks to see whether those are appropriate storage for water, if they have a lid, if there are larvae, if there are flower vases with larvae, all of that. And they [the community] receive explanations, that you have to throw those away, that it's better to have your flowers but with wet earth and all of that, to avoid mosquito propagation. We also tell them to throw away all trash materials because that's where the mosquito lives. Health professionals advocated including Zika virus as part of ongoing community visits that all clinic personnel have to fulfill. This was piloted when Zika virus cases first appeared and could continue:Midwife 4When they come to the prenatal control that makes things easier for when she comes in for her method because she already talked to him, and he already hears about it. When he comes, in some cases husbands come, we take advantage and we talk about everything because it's an opportunity. We think [it has to be now because] “When will he come back? IF he comes back.” And I'm very happy because there's many that come, maybe late in the day or on their off days from work they come with their wives. Midwives also wanted to establish a way to have male partners participate in prenatal visits. Some do participate and midwives reported that this is an excellent space to engage men in discussions of sexual transmission of Zika virus, the need for condom use during pregnancy, and future contraceptive usage.3.7Nurse 5So, there was some training, but really who got it, the midwives! We got some leaflets and condoms. Nurse 4What I think is that [midwives] who have been trained should do replicas to all others. Here we didn't do it. They shared the materials but the proper training we did not get. As explained in the referral system section, the lack of counter‐referral protocols limits the ability of midwives and nurses to follow‐up suspected Zika virus cases. This poses problems for effective CZS surveillance. While nurses see a greater role for themselves in managing and treating the effects of Zika virus in children, they have not been trained to do it:Nurse 3For newborns, it's four visits the first month. One each week. And there we observe, we measure head circumference, abdominal, thoracic, we evaluate the child completely. Up until two months. Until the second month we measure head circumference. Nurses’ current lack of knowledge about CZS symptoms limits their ability to identify cases of CZS. Currently, most only identify microcephaly as a sign of the syndrome and focus only on head circumference. Even though they evaluate other developmental milestones, none mentioned other CZS symptoms:Nurse 5Something we have reinstated a lot since last year is the head circumference measurement. When I returned, I came in November 2016, I didn't see that it was done in our well‐child care. I insisted with the person in charge at the time, that it was a very important step in the child's evaluation, that we could not avoid it. They argued that there was no proper measuring tape for true head circumference measurement, the one that comes from the Ministry [of health], there wasn't even a simple measuring tape, but I thought there were ways to get it. Then we got one and from there we have been doing it, it's one of the things we do to avoid [Zika], to go on measuring, comparing with the baseline of head circumference at birth, with its age and to compare with the growth charts and see if it is in normal range. However, proper training and tools to appropriately measure head circumference are also needed:In addition to pointing out limitations of the tools to monitor development, this nurse also explained measuring and identifying problems in head circumference growth as mechanisms to “avoid” Zika or its effects in children. It further reflects the existing misunderstanding about CZS, the impact of Zika virus, and opens up a discussion about the value of interventions to prevent and cure health problems over those of monitoring, identifying, and dealing with permanent/chronic health conditions.Nurse 3What we do is TEPSI, which is a cognitive test. We do it with children who are two, three years old. We have a little book where they can identify animals, we ask them their name, how old they are. When asked about their capacities to include any screening for neurological and cognitive problems as part of child growth and development care, nurses only mentioned one test they apply to children for psychomotor development (TEPSI) between 2 and 5 years.Overall, nurses would like to receive more training about Zika virus, but mainly see a role for themselves in CZS management.4Effective long‐term prevention of Zika virus, including management of CZS, poses challenges for health policy and prevention strategies. Moving from early response to Zika virus infection to endemic scenarios will require coordinated policies that engage all primary care personnel.Studies have shown that health personnel in Zika endemic areas have a high level of awareness, but a low level of knowledge and preventive practices related to Zika virus preventionAs in many countries, the focus of early Zika virus prevention was pregnant women. Midwives have easily incorporated and maintained Zika virus related counseling into their prenatal care work but have yet to incorporate it as part family planning and contraceptive care. Despite concerns about fetal effects, both nurses and midwives have limited knowledge about CZS. They only associate it with microcephaly, although the full spectrum of CZS includes epilepsy, cerebral palsy, delayed development of language and/or motor skills, strabismus, visual abnormalities, and heart, renal, and urinary tract disorders, among others.Midwives and nurses pointed to the absence of microcephaly as a mark of the relatively benign nature of Zika virus infection in Piura, especially compared with the deaths caused by dengue. However, the absence of microcephaly cases in official records does not necessarily mean that there are no cases, especially with the technical limitations described above and in the literature.Women of reproductive age, who should be well aware of the risks posed by Zika virus when considering their family planning options, have not been consistently targeted either. This reflects the limitation of the primary health system to reach women of reproductive age before they are pregnant and access prenatal visits. This also reflects the political reticence around openly discussing and fulfilling women's sexual and reproductive rights and needs, including offering culturally acceptable contraception.The siloed organization of health programs and targeted populations has also been challenged during the Zika virus epidemic. The few suspected cases of newborns with anomalies required the exchange of medical information between nurses, midwives, and doctors at the primary care level, prior to their referral for confirmation, and between different levels of care, which was cumbersome, slow, and inconsistent. Lack of a counter‐referral system in place poses a challenge to children's health and CZS surveillance at primary care level.The occurrence of CZS also revealed the conditions and limitations in which standard child growth and development services are provided, and the challenges they present to identify and care for the wider spectrum of symptoms and conditions that might not present immediately or at birth. Constant follow‐up is crucial for early identification of suspected cases and remedial actions at the primary care level provided by local nurses familiar with the child during his/her first years of life.Finally, it is important to acknowledge that health personnel at primary care centers share cultural values and practices and risk perceptions with the community they are part of and the population they serve. Their relatively low perception of Zika virus risk may reflect and reinforce local attitudes.The present study is novel in its approach to trying to understand how health providers view themselves within Zika virus related care. However, it has some intrinsic limitations. Firstly, flooding and a dengue epidemic disrupted data collection and moved all local resources toward dengue prevention. This may have influenced the conflation between dengue and Zika virus symptoms in the area. Additionally, this was a qualitative, small sample study that may inform future research and questions but is not meant to be generalized.In conclusion, in an endemic scenario of long‐term surveillance and management of Zika virus and CZS, diagnostic tools, training, and surveillance resources need to be increased to be effective. A cost‐effective and culturally competent approach should likely include a broader focus on family planning and child development surveillance. This supposes cross‐disciplinary collaboration between nurses and midwives. The present findings are important for health policymakers who have to design training, surveillance, and referral processes at regional and local levels.RI and LG conceived and designed the study, conducted data analysis, and drafted the manuscript. RI conducted the interviews and fieldwork. All authors contributed to the revision of the article and approved the final version of the manuscript.The authors have no conflicts of interest.