This prospective cohort study sought to estimate health system and household costs for episodes of diarrhoeal illness in Malawi.Data were collected in two Malawian settings: a rural health centre in Chilumba and an urban tertiary care hospital in Blantyre.Children under 5 years of age presenting with diarrhoeal disease between 1 January 2013 and 21 November 2014 were eligible for inclusion. Illnesses attributed to other underlying causes were excluded, as were illnesses commencing more than 2 weeks prior to presentation. Complete data were collected on 514 cases at both the time of the initial visit to the participating healthcare facility and 6 weeks after discharge.The primary outcome measure was the total cost of an episode of illness. Costs to the health system were gathered from chart review (drugs and diagnostics) and actual hospital expenditure (staff and facility costs). Household costs, including lost income, were obtained by interview with the parents/guardians of patients.Total costs in 2014 US$ for rural inpatient, rural outpatient, urban inpatient and urban outpatient were $65.33, $8.89, $60.23 and $14.51, respectively (excluding lost income). Mean household contributions to these costs were 15.8%, 9.8%, 21.3% and 50.6%.This study found significant financial burden from childhood diarrhoeal disease to the healthcare system and to households. The latter face the risk of consequent impoverishment, as the study demonstrates how the costs of seeking treatment bring the income of the majority of families in all income strata below the national poverty line in the month of illness. We collected costs of illness directly from patient caregivers and clinic records rather than relying on model-derived estimates.Collection of information on lost income means that we were able to provide more holistic measures of the effect of illness on household economy.Only two clinics were sampled, so the conclusions’ generalisability to other locations in Malawi is uncertain.Estimates of the contribution of previous visits to overall costs are likely to be confounded by selection bias, as the most severe patients are likely to have been seen before and to have higher costs.Diarrhoea is the major cause of childhood illness and death. It is responsible for an estimated 9% of all under-five deaths globally,In addition to the burden of disease and death in children, treatment costs can cause significant financial strain to households and healthcare systems alike. In order to understand comprehensively the impact of diarrhoeal disease and the potential economic benefit of vaccines, one must examine the actual costs of care incurred by the healthcare system and by households.Most economic evaluations have relied on model-derived cost estimates based on extant, retrospective data. Very few studies have prospectively collected direct individual patient-level costs of diarrhoeal illness at both inpatient and outpatient settings.In this post hoc analysis of data collected for a cost-effectiveness analysis,This prospective cohort study was conducted in Malawi, a country of approximately 16 million people in southern Africa. Its per capita gross national income of US$350 places it 210 of 216 countries and territories ranked by the World Bank.The study was conducted at two sites: Queen Elizabeth Central Hospital (QECH), a national referral hospital that provides both inpatient and outpatient care, and Chilumba Rural Hospital (CRH), which provides outpatient primary care for children under 5 years and accepts inpatients, although generally low acuity cases. QECH is located in Blantyre, a district of approximately 1.3 million in Malawi’s Southern Region. It receives approximately 90 000 presentations per year, 25 000 of which are admitted to the inpatient facility. CRH is located in Karonga, a district of approximately 200 000 in the Northern Region. Chilumba hosts a demographic surveillance site with a population approaching 40 000.Eligible participants were children under 5 years of age living in Blantyre district or within the demographic surveillance population in Chilumba and presenting with diarrhoea at either facility during the study period (1 January 2013 to 21 November 2014). Illnesses attributed to other underlying causes were excluded, as were illnesses commencing more than 2 weeks prior to presentation. In addition to identifying participants at registration/triage, admission logs and electronic records (at QECH only) were reviewed for missing cases.After identifying each participant and obtaining consent from their caregiver, a trained research assistant administered a standard case report form to the caregiver. The questionnaire included demographic information, socioeconomic status and the history of the child’s illness prior to presentation at the facility, as well as all illness-associated costs incurred prior to presenting at the facility. Daily review of medical charts was undertaken during admission for inpatients to gather information on drugs dispensed, and laboratory investigations or procedures performed. All participants’ homes were visited by research staff 6 weeks postdischarge to evaluate whether any subsequent illness-related costs were incurred by the household.Pretreatment costs included direct medical costs paid by the household for care sought prior to the date of the index visit, which we defined as the date of an outpatient visit at either facility or the first day of an inpatient stay. Since the health facilities at which previous care was sought were not study sites, costs to the health system were not included in the pre-index costs.Costs of the index visit were divided into health system and household costs. Costs to the health system included drugs, laboratory investigations, staff and facility costs such as laundry, kitchen, sanitation and security. The cost of drugs was calculated based on acquisition costs for drugs used at the index visit, with the quantity of injectable drugs being rounded up to the nearest full phial size. Investigations performed were costed on charge per test, except for those performed as part of clinical research, which were excluded. Staff costs for inpatient visits used the combined daily salary of all staff present divided by the number of beds in the ward, then multiplied by the patient’s length of stay in days; staff costs of emergency outpatient visits were calculated by dividing the combined daily salary of staff by the mean daily number of visits to the department. Facility costs were calculated by dividing the facility’s mean daily cost for services and subsistence goods by the number of hospital beds, then multiplying by the patient’s length of stay. The cost of consumable goods in the clinic such as tubing for intravenous drips was not included. Household costs included transportation to the index visit for the patient, their main caretaker and any other household visitors during admission; direct medical costs ; lost income attributable to the episode of disease; and subsistence (food and shelter) during the visit. Lost income was calculated by dividing the self-reported monthly income by the number of days in the month, then multiplying by the number of days spent in care taking for this episode of illness.Ongoing costs were assessed at a follow-up visit to the patient’s home 6 weeks after facility discharge. Costs related to any long-term disability caused by the episode were not included among these ongoing costs. Again, costs to the health system for further treatment could not be ascertained due to other sites’ being outside the network of research sites.All costs were collected in Malawi kwacha and converted to US$ based on the mid-market exchange rate per the Malawi Reserve Bank on 15 July 2014.The study’s minimum sample size of 88 was calculated to provide an estimate of household costs with a margin of error ≤10% given a coefficient of variation of 0.5.Income quintiles were calculated based on self-reported monthly income within the sample itself—that is to say that a household labelled as belonging in the highest income quintile is within the top 20% of household incomes in this sample. Using one-way analysis of variance (ANOVA), we tested for differences among income quintiles in mean overall household costs at each geographic and treatment setting.2 test to compare the proportion of patients with prior/ongoing costs in different settings, and a rank-sum test to assess differences in component costs between these groups. We also used rank-sum tests to assess the significance of differences in total  and household cost between geographic and treatment settings.To explore associations proposed in previous research between prior treatment and cost, we constructed a generalised linear model of total costs and household costs with log link on gamma distributional family, adjusted for length of stay, geographic setting and treatment setting. We used a ΧEthical approval was provided by the National Health Sciences Research Committee, Lilongwe, Malawi (1073), and by the Research Ethics Committee of the University of Liverpool, UK (000490). Written consent was obtained from the parents or guardians of participating children.A total of 529 individuals participated in this study, although 15 were excluded due to incomplete data see . The majTotal costs for the episode of illness and costs by payer are reported in We performed a sensitivity analysis to assess the potential impact to overall costs at the index visit if the 15 dropped observations were included. Were these costs representative of the 25th percentile of costs, mean overall costs would decrease from $39.15 to $38.32; were they in the 75th percentile of costs, mean overall costs would rise to $39.44. This would result in a 2% decrease in total costs, or a 0.6% increase, respectively, compared with the weighted average of costs across all visit types.For inpatient settings, staff and facility costs contribute the most to health system costs see . These tLost income and direct medical expenses make up the majority of household costs for all except rural, outpatient visits, whose costs are dominated by transportation and lost income see . Of patiUsing one-way ANOVA to detect differences in mean household expenditure (exclusive of lost income) between income quintiles, we found a significant difference only in urban, inpatient encounters see . The higCosts to the households of patients for whom previous treatment was sought were 2.04 times higher (p<0.001) once adjusted for length of stay, geography and treatment setting; overall costs were 1.25 times higher (p=0.016). This difference in overall costs is driven in part by spending prior to the index visit, which averaged between $0.92 for rural, outpatient index visits and $3.01 for rural, inpatient index visits. A rank-sum test on transportation costs by prior treatment status showed significantly higher transportation costs for this group (p<0.001), suggesting the source of these differences. Median monthly income for the group who sought prior treatment was higher than the group who did not, at $34.09 and $22.73, respectively. A rank-sum test again showed a significant difference in both monthly income and lost income due to the illness (length of stay was not significantly different between the groups). We repeated the rank-sum test looking for variation in transportation costs during the rainy seasons (November through April) of those who received prior treatment, but this test’s results were not significant.Even following the introduction of the rotavirus vaccine, diarrhoeal disease continues to be a significant burden on the world and on low-income countries in particular. While deaths among African children due to diarrhoea declined 43% between 2000 and 2013,This study focused on the economic burden of these diseases by combining costs ascertained through both interviews and chart review. It found significant household costs primarily in the inpatient and urban settings with lost income and direct medical costs dominating the contributing costs. Even rural, outpatient visits can be financially problematic, however, if they are frequent, or if the family is especially vulnerable. Household costs for the episode of illness (excluding lost income) exceeded monthly household income in 8% of cases treated inpatient and 3% of cases treated outpatient; including lost income, these numbers rise to 17% and 9%, respectively.Taking into account lost income, mean household costs for patients seen in the rural health centre were $19.16 and $1.81 for inpatient and outpatient treatment, while patients seen at the urban hospital had mean household costs of $25.36 and $15.48 for inpatient and outpatient treatment. The high cost of inpatient treatment in the urban setting likely reflects the higher acuity of illness at that setting: the mean Vesikari score of study participants seen in QECH’s inpatient ward was 11, which indicates severe disease. Rural inpatients, by comparison, had a mean Vesikari score of 6.Putting household expenditures in context, 72.2% of the Malawian population lives on <$1.25 per person per day.The results of our study can be compared with several other studies of the economic burden of diarrhoeal disease in the low-income, sub-Saharan context. An analysis of economic data from the Global Multicenter Enteric Study found mean household costs of $2.77, $6.57 and $4.33 (in 2014 USD) for Gambia, Kenya and Mali, respectively, for a representative sample of inpatient and outpatient medical encounters, not including lost income.When costs are analysed by income quintile, mean household expenditure by quintile is only significantly different in the urban, inpatient setting. This demonstrates that the poorest segments of Malawian society who attend public healthcare facilities generally incur the same costs as their wealthier peers in the same settings. This finding is supported by other evidence from Malawi which found that care-seeking behaviour for children with diarrhoea does not vary by income level.The pattern of identical expenditure across income quintiles in most settings means that the poorest households expose themselves to greater risk of impoverishment by seeking treatment see . After aWe are left to explain why spending increases with income quintile in the urban inpatient setting see . One expOne driver of high overall costs in the urban setting at large may be the seeking of prior care. Of patients seen in the urban setting, 35% had been seen at a prior healthcare facility. Testing the relationship between prior care and income quintile using a logistic regression does not suggest a significant relationship between the two, whether income quintile is represented as a continuous or factor variable. Excluding lost wages, household costs for cases in which prior care is sought cost twice as much as cases in which the index visit is the first encounter with the healthcare system during this episode of illness. Transportation costs are the likely source of difference, suggesting that patients who sought treatment prior to the index visit come to the hospital either from farther away or more urgently than those for whom the index visit was their first visit related to this episode. Transportation costs predominate costs accrued prior to the index visit as well, accounting for 62%; spending at health centres and pharmacies accounted for 13% and 11%, respectively. Selection bias likely plays a role as well, as the most seriously ill patients are likely both to be seen multiple times and to incur greater overall costs.The conclusions in this study are limited by its being a post hoc, unpowered analysis of data gathered for a cost-effectiveness study. That is, the sample size of the study was not powered to detect differences between treatment settings, geography or any of the other characteristics examined in our regression analysis. The study’s generalisability may also be limited due to the data collection’s having taken place in two locations that represent extremes of acuity and population density within the Malawian healthcare system. We cannot rule out greater geographic variation in financial outcomes than our study results suggest.Future work in this area should focus on the long-term consequences to the household of financial shock associated with an episode of diarrhoeal disease, or with repeated diarrhoea due to undernourishment and other risk factors. Especially of interest is the long-term loss of economic potential due to childhood illness: if a child is frequently or severely ill, health-related expenditures, including time needed for caretaking, may preclude investment in other areas that could improve the financial situation of a household.This study has evaluated directly the cost of 514 episodes of childhood diarrhoeal disease to both the households and health system of Malawi. It found that households bear a substantial fraction of the total costs of an episode of illness and that there is a considerable risk of impoverishment due to severe or repeated episodes of diarrhoeal disease.The conclusions of this study are broadly applicable in the sub-Saharan setting—especially to those countries where the rotavirus vaccine is widely used—regardless of whether healthcare is provided to the public for free or through an insurance programme. In the latter case, only direct medical costs to the household would be covered. Lost income, transportation and ongoing costs of care were all found in this study to impose substantial financial burdens, and this observation would not be changed by the use of health insurance.This study’s findings can be applied to cost-effectiveness analyses of enteric vaccines and other means of preventing the transmission of diarrhoeal disease and can be used by healthcare planners to help prioritise interventions. By providing data on the lost financial opportunity caused by an episode of diarrhoeal disease, this study allows for a more precise accounting of how an averted episode of diarrhoeal disease should be valued.


Culicoides paraensis. Currently, there is no evidence of direct human-to-human OROV transmission. OROV fever is usually either undiagnosed due to its mild, self-limited manifestations or misdiagnosed because its clinical characteristics are similar to dengue, chikungunya, Zika and yellow fever, including malaria as well. At present, there is no specific antiviral treatment, and in the absence of a vaccine for effective prophylaxis of human populations in endemic areas, the disease prevention relies solely on vector control strategies and personal protection measures. OROV fever is considered to have the potential to spread across the American continent and under favorable climatic conditions may expand its geographic distribution to other continents. In view of OROV’s emergence, increased interest for formerly neglected tropical diseases and within the One Health concept, the existing knowledge and gaps of knowledge on OROV fever are reviewed.Oropouche fever is an emerging zoonotic disease caused by Oropouche virus (OROV), an arthropod transmitted Orthobunyavirus circulating in South and Central America. During the last 60 years, more than 30 epidemics and over half a million clinical cases attributed to OROV infection have been reported in Brazil, Peru, Panama, Trinidad and Tobago. OROV fever is considered the second most frequent arboviral febrile disease in Brazil after dengue fever. OROV is transmitted through both urban and sylvatic transmission cycles, with the primary vector in the urban cycle being the anthropophilic biting midge  Peribunyaviridae family , which is transmitted to humans predominantly by the biting midge Culicoides paraensis [Oropouche fever is an emerging zoonotic disease caused by Oropouche virus (OROV), an arbovirus of the Orthobunyavirus genus in the araensis ,2. OROV araensis ,4. OROV OROV is a member of the Orthobunyavirus genus that comprises more than 170 viruses of 19 different serogroups and 48 species complexes , includiOne of the important characteristics of the Simbu serogroup viruses is their high genetic diversity, which is attributed to their wide geographical distribution. According to recent studies, this trait is most likely associated with divergences in either the vectors or the definitive hosts, or in both, underpinned by variations in the M segment of these viruses due to re-assortment or adaptation to diverse environments ,6,7. Of OROV is a negative-sense, single-stranded RNA virus with a spherical lipid-enveloped genome, sized from 80 to 120 nm in diameter, consisting of three single stranded RNA segments called large (L), medium (M) and small (S), surrounded by helicoidal nucleocapsid and encoding the viral RNA-dependent RNA polymerase (RdRp), the viral surface glycoproteins (Gn and Gc) and the nucleocapsid protein (N), respectively [Through phylogenetic analyses, three virus genotypes  were identified in 2000, from Brazil, Trinidad, Peru and Panama, respectively , and, teThe high levels of genetic similarity observed in members of the OROV species are associated to re-assortment events amongst these species, occurring during many occasions of host and vector range changes, and pathogenicity variations . Re-assoC. paraensis belongs to genus Culicoides , which includes over 1400 species distributed worldwide, except the polar regions and New Zealand [Culicoides includes 32 subgenera and 38 groups, yet 13% of the species remain unclassified [Culicoides genus is of major international public health interest because it includes species that are vectors of arboviruses of human and veterinary importance; the majority (96%) of these species are hematophagous, feeding on humans and other mammals such as sheep, goats, cattle, horses, deer, and birds [Culicoides midges worldwide, many of them of major veterinary interest such as the Bluetongue virus (BTV), Equine encephalitis virus (EEV), Epizootic hemorrhagic disease virus (EHDV), Akabane virus (AKAV), Bovine ephemeral fever virus (BEFV), African horse-sickness virus (AHSV), Palyam viruses and Schmallenberg virus [OROV’s predominant vector  Zealand . Culicoiassified . The Culnd birds ,18. Morerg virus ,20,21. TCulicoides midges remains to be determined, but existing data indicate that they are mainly mammalophilic and/or ornithophilic, blood-feeding on various mammals and birds, depending on the availability of these hosts [Culicoides midges have been investigated using either laboratory methods such as serological analysis of abdominal blood by precipitin test, enzyme linked serological assays (ELISA) and polymerase chain reaction (PCR) or observational studies based on collection of adult female midges by light, sticky or animal-baited traps or by direct aspiration from animals, the latter being the most credible study method [Culicoides species can be opportunistic in host preference [Culicoides related to larval stages remains unexplored. Culicoides breed in habitats that provide the proper moist environment for the eggs to survive and hatch, like mud at the soil-water interface, dung pats, highly organic and moist soil substrates such as manure mixed with soil, which can accommodate abundant vectors very close to livestock, thus facilitating arbovirus transmission to animals and to humans living in the proximity of animal farms [The actual host range of se hosts . The hosy method . Quantiteference . Only feeference ,23. Eggsal farms .C. paraensis is reported to be abundant during the hot or rainy months of the year and is characterized by broad geographic distribution, a feature attributed to its ability to survive and reproduce in semi-urban regions that are close to areas of high human density [C. paraensis develop in various habitats capable of remaining moist enhancing larvae feeding in dry periods, like rainforests, riverbanks, damp soil, tree holes, decaying organic matter of plant origin such as rotting banana and plantain stalks, stumps and cacao husks [C. paraensis prefer biting indoors during day and night, more often by late afternoon, with a reported indoor/outdoor ratio of 29% [C. paraensis bites have been reported to be implicated in common allergic reactions and skin irritation, like dermatitis, eczema or scars [ density ,25,26,27ao husks ,29,30. Co of 29% , during or scars .Culex quinquefasciatus, Coquillettidia venezuelensis, and Aedes (Ochlerotatus) serratus can breed and may be naturally infected by the virus [Cq. venezuelensis, and Ae. serratus in the Trinidad and Amazonian region of Brazil, respectively [Cq. venezuelensis is reported to be breeding in aquatic ecosystems, such as pools, lakes and rivers, characterized by nocturnal biting activity [Ae. serratus demonstrates diurnal biting activity, feeding preferably from large mammals [Cx. quinquefasciatus (member of Cx. pipiens complex) commonly known as “southern house mosquito”, is one of the most widespread mosquitoes in tropical and subtropical areas, feeding in both animals and humans, occurring most frequently in urban environments [OROV is also distributed to areas where hematophagous mosquito species he virus ,32,33. Iectively . Both spectively ,35. Cq.  mammals . Cx. quironments . It is cronments ,36, and ronments .C. paraensis is the primary vector [C. paraensis as a main urban vector has been supported by experimental and epidemiological data. Pinheiro and colleagues proved that C. paraensis midges are able to transmit OROV to hamsters 6 to 12 days after blood-feeding on viremic patients, and the threshold titer enabling transmission and infection was approximately 5.3 log10 SMLD50/mL [Culicidae species, such as Cx. quinquefasciatus can contribute to the OROV transmission, but relevant experimental studies show the threshold of infection for this species to be high (≥9.5 log10 SMLD50/mL) indicating low efficiency of virus transmission by this mosquito species [C. paraensis efficiency to transmit the disease feeding on lower virus-titer blood-meals provides strong evidence that they are the most important OROV vectors [Cx. quinquefasciatus is recognized to be an anthropophilic vector to a lesser extent, OROV is the only member of the Orthobunyavirus genus isolated from this mosquito species, based on the detection of OROV’s SRNA in both patients and insects by nested-reverse transcription-polymerase chain reaction (RT-PCR) [OROV is conserved in nature by an urban cycle and a sylvatic cycle, which may include several different vectors . In the y vector , which hy vector . The rolMLD50/mL . It has  species . The C.  vectors . Althoug(RT-PCR) . The epi(RT-PCR)  and duriBradypus tridactylus), nonhuman primates such as capuchin monkeys (Sarajus spp.), black-and-gold howler monkeys (Alouatta caraya), black-tufted marmosets , rodents (Proechimys spp.), and birds  implicated in the transmission of OROV. Nevertheless, the existing literature on OROV detection in the reservoir hosts is rather limited. Published data report OROV isolation from one sloth in 1960 (Brazil), antibodies detection in one rodent, 34 wild birds and 12 domestic birds during an epidemic in Mojui dos Campos, Pará State, Brazil in 1975 [Cq. venezuelensis, Ae. serratus, Cx. quinquefasciatus and midges of the genus Culicoides have been reported to be likely vectors in the sylvatic cycle as indicated by epidemiological and laboratory based evidence [In the sylvatic cycle mammals, wild and domestic birds are the natural reservoir hosts. Antibodies to OROV have been found in pale-throated three-toed sloths  and from Ae. serratus mosquitoes in Brazil, both hosts being captured near a forested area during the construction of the Belém-Brasilia highway [C. paraensis and Cx. quinquefasciatus were the most common vectors in the endemic areas [The epidemiological history of OROV fever demonstrates the role of human-induced environmental and climatic changes on hosts’ and vectors’ spread and disease prevalence. For the first time, OROV was isolated in 1955 from a febrile human case in the island of Trinidad in the Caribbean Sea; the infected patient was a forest worker and during that incident neutralizing antibodies were detected in the blood of a few more forest workers and from a number of cebus and howler monkeys that were living in Trinidad [ highway . Within  highway ,32,39. I highway ,41. It iic areas , which iic areas .Callithrix sp., in southeastern Brazil , demonstrated the OROV’s potential to spread to new hosts and new areas [From 1980 to 2005, only sporadic cases or self-limited outbreaks were reported in the Brazilian Amazon regions, mainly in small villages indicating potential silent circulation of the virus . In mostew areas . OROVs eIn 2003–2004, two small outbreaks of OROV fever occurred in urban and rural municipalities of Pará State . In 2006, a large epidemic  was recorded in two more municipalities  signifying re-emergence of OROV in Pará state after 26 years. The observed epidemic gap was attributed to possible accumulation of OROV susceptible people not previously exposed to the virus . Serological surveys performed among febrile patients in Parauapebas and Porto de Moz revealed that 20.7% and 46.7%, respectively, had HI antibodies and 22.3% and 52.1%, respectively, had IgM antibodies to OROV . A similC. paraensis from South and Central America to northern United States of America, coupled with anthropogenic environmental disturbances and extensive human and animal travelling accentuate the potential for OROV to emerge in other territories. From the reviewed literature on OROV fever emergence in Brazil, it is clear that the common features of the affected areas were the tropical climate, high frequency of rains, similar activities of the residents , unplanned urbanization and very poor standards of living [In 2007–2008, OROV outbreaks were recorded in Manaus and infection was detected by an in-house enzyme immunoassay for IgM antibodies in acute-phase serum samples, using infected cell culture as antigen (EIA-ICC) . In 2011f living ,28,30,57The first isolation of OROV virus in Panama was recorded in 1989 in the village of Bejuco from febrile patients suspected of dengue fever, during a dengue surveillance program in the greater metropolitan area of the Panama City. At that time, OROV was not known to exist in Panama, but a later retrospective serological study of sera collected in 1968 and 1978 from residents of the affected community revealed that 25% of the samples had OROV antibodies, suggesting earlier human exposure to virus and possibly infection, which had gone unrecognized ,43.In 1992, a couple of years after OROV fever was detected in Panama, the disease was detected in Peru, involving five febrile patients living in Iquitos port on Amazon River , and sinRecent studies based on epidemiological surveillance in humans and wild mammals have demonstrated circulation of OROV in Argentina, Bolivia, Colombia, Ecuador and Venezuela ,61,62,63Mesocricetus auratus), in which both the virus replication and the disease may occur, has been used for subcutaneous experimental inoculation of OROV by Rodrigues et al. (2011) [It is recognized that OROV is highly detectable in blood from the onset of infection, and can progressively reach the neural routes, resulting in a systemic infection and primary central nervous system (CNS) inflammatory response . Since t. (2011) . Hamster. (2011) ,67. In t. (2011) . The hep. (2011) ,66. It h. (2011) .Experimental infections in mice demonstrated severe manifestations of encephalitis related to extended spread of OROV through the brain parenchyma. In one study, suckling BALB/c mice were subcutaneously inoculated with OROV developing severe disease five days post inoculation with lethargy and paralysis leading to death >80% of the animals within 10 days. Viral replication in brain neurons pointing out the neurotropism of OROV, was documented by in situ hybridization, immunohistochemistry and virus titration . DespiteActivation of mitochondrial antiviral-signaling protein (MAVS), interferon-regulatory factors (IRF) 3 and 7, and production of type I interferon (IFN-I) have been found to play an important role in the innate immune system’s response to viral infection, by controlling the virus replication, the liver damage and the progressive death in mice experimental models . In addiOROV fever is manifesting as a self-limited, dengue-like, acute febrile illness that lasts 2–7 days, and is associated with a variety of symptoms such as fever, chills, headache, myalgia, arthralgia, malaise, dizziness, nausea, vomiting, photophobia, retro-ocular pain, and, in rare occasions, skin rash appearing more commonly on trunk and arms, hemorrhagic signs such as spontaneous bleeding, petechiae, epistaxis, gingival bleeding, and CNS signs like aseptic meningitis or meningoencephalitis ,47,50,53The most frequently reported symptoms as recorded during large epidemics in Brazil were: fever (100%), headache 79.3%), arthralgia (68.7%), myalgia (30%) in Parauapebas and Porto de Moz, (2003–2004) outbreak , fever , HI test ,60,71,74The HI test is considered to have higher sensitivity but lower specificity when compared to other serological assays such as MAC Elisa, but specific antibodies can be detectable for long periods after infection . Enzyme Molecular techniques, such as nested RT-PCR, have been successfully applied as important diagnostic tools, for rapid and specific diagnosis of OROV infection in both serum and CSF samples ,73. A moCurrently, the available treatment for OROV infection is symptomatic, which means that the provided medication is only easing the symptoms but is not affecting the causative agent (by killing the virus or inhibiting its replication). Generally, the prognosis for OROV fever is good and no deaths have been reported ever since the first outbreak in 1960. In the existing literature, data on in vitro or in vivo experiments for potential treatment or prevention options are scanty. A study by Livonesi et al. (2006) on ribavirin, a broad-spectrum antiviral drug, reports no inhibitory effect on OROV and another study by Livonesi et al. (2007) reports some antiviral activity of IFN-α on OROV ,79. ConcN,N-Diethyl-meta-toluamide (DEET) have demonstrated very sufficient insecticide effects against Culicoides species. However, the ecological consequences of the insecticides usage at a large scale are of particular concern [Culicoides populations. Education of human populations in endemic regions  about the seasons of high exposure risk, the potential implications on human health due to midge biting and personal protection options can contribute significantly to OROV fever prevention.Chemical insecticides such as deltamethrin and  concern ,82. Alte concern ,82. The OROV fever is an emergent zoonotic disease that is currently endemic in certain regions of South and Central America. Despite the fact that it has been infecting over half of million people in South America, the disease—like most of the arboviral diseases—was a neglected disease for almost sixty years. Thus, data on the distribution, prevalence and incidence rates in human, animal and vector populations are inadequate and possibly underestimated. OROV fever is an acute febrile illness, but is self-limited and it is not a fatal disease, which may explain the lack of adequate attention by researchers, international health agencies and policy makers. OROV’s spread is related to environmental factors including climate, fauna and flora changes, as well as to socioeconomic factors such as human population demographics, agricultural practices and poor standards of living. There are contradictory reports on the possible impact of age and gender on the prevalence of the disease and there are no reports relating the disease severity with factors such as the geographic region, season, vector species or the virus lineage and/or genotype. Nevertheless, there is full agreement of the literature on the factors incriminated in OROV disease emergence, which are summarized in: deforestation, human population growth, intensive animal farming, monoculture plantations, highway and dam building in the tropics, climate change, increased human and animal mobilization, and globalization of food trade. The OROV fever emergence and distribution, like other vector-borne diseases, is a consequence of the ecologic equilibrium’s disturbance, which is observed world-wide. Considering that environmental, climate and demographic changes are a global phenomenon, it will not be surprising for OROV to spread outside Latin America in the near future. Furthermore, taking into account that this is another emerging zoonotic disease, it has to be encountered within the “One Health” approach through the collaboration of human and veterinary medicine, entomology, biology and environmental sciences.


During 2013–2014, French Polynesia experienced an outbreak of Zika virus infection. Serosurveys conducted at the end of the outbreak and 18 months later showed lower than expected disease prevalence rates (49%) and asymptomatic:symptomatic case ratios (1:1) in the general population but significantly different prevalence rates (66%) and asymptomatic:symptomatic ratios (1:2) in schoolchildren. Flaviviridae, genus Flavivirus), an arthropodborne pathogen, is transmitted to humans by Aedes spp. mosquitoes, but nonvectorborne transmission  has also been reported . During February and March 2014, we conducted a cluster sampling among the general population living in the 5 archipelagos. We randomly recruited a total of 196 participants on the most inhabited islands of each archipelago: Tahiti and Moorea (Society), Rangiroa and Makemo (Tuamotu), Nuku Hiva and Hiva Oa (Marquesas), Rurutu , and Rikitea (Gambier) . The population of ≈270,000 inhabitants lives on 74 islands  . BecauseAll participants were asked to declare whether they had clinical manifestations suggestive of past Zika infection. Adults provided written informed consent before enrollment, and parents or guardians gave consent for their children. Participants’ blood samples and personal data were anonymized before processing, and the study was approved by the Ethics Committee of French Polynesia (no. 60/CEPF 06/27/2013).We used a recombinant antigen–based indirect ELISA to detect Zika virus IgG in blood samples collected in 2014 from the general population and schoolchildren  and 43% (95% CI 33%–53%), respectively, for participants from the general population sampled in 2014 and 66% (95% CI 60%–71%) and 29% (95% CI 24%–34%), respectively, for schoolchildren sampled in 2014 . SeropreDuring the October 2013–April 2014 Zika infection outbreak in French Polynesia, ≈11.5% of the population sought medical care for symptoms suggestive of Zika infection  and 53% (95% CI 45%–61%), respectively, not substantially different from those during the first cluster sampling in the same islands . The finding that the Zika virus seroprevalence rate did not increase between the 2 sampling periods suggests that the virus did not actively circulate after the end of the outbreak. In contrast, the decrease in the Zika virus seroprevalence rate, even if not significant, suggests that Zika virus IgG titers may drop over time.Within 2 months after the end of the outbreak, the Zika virus seroprevalence rate among schoolchildren (6–16 [median 11] years of age) on Tahiti was substantially higher than that among the general population (4–88 [median 43] years of age) . In contrast, the proportion of asymptomatic Zika virus infections was substantially lower among schoolchildren (29% [95% CI 24%–34%]) than among the general population (53% [95% CI 45%–61%]). Dengue virus (DENV) may provide cross-protection against Zika infection; thus, the higher DENV IgG seroprevalence among adults may explain why fewer adults than children were infected by Zika virus (In the 3 groups tested, no difference was seen by sex in the seroprevalence rate or the proportion of asymptomatic infections. However, because the sampling scheme was not initially designed to compare data by sexes, data could not be extrapolated to the population level.Our findings show that <50% of the population of French Polynesia had detectable Zika virus IgG. This seroprevalence rate is much lower than the 86% attack rate estimated by Kucharski et al. (


Seafarers sail the high seas around the globe. In case of illness, they are protected by international regulations stating that the employers must pay all expenses in relation to repatriation, but very little is known about the cost of these repatriations. The objective of this study was to estimate the financial burden of repatriations in case of illness. We applied a local approach, a micro-costing method, with an employer perspective using four case vignettes: I) Acute myocardial infarction (AMI), II) Malignant hypertension, III) Appendicitis and IV) Malaria. Direct cost data were derived from the Danish Maritime Authority while for indirect costs estimations were applied using the friction cost approach. The average total costs of repatriation varied for the four case vignettes; AMI , Malignant hypertension , Appendicitis  and Malaria  mainly due to large variations in the average direct costs which ranged between 9560 euro in the malaria case and 77,255 in the AMI case. Repatriating an ill seafarer is a costly operation and employers have a financial interest in promoting the health of seafarers by introducing or further strengthen cost-effective prevention programs and hereby reducing the number of repatriations. Seafarers are an essential workforce to the global economy with around 1.5 million people working day and night , 3. The Limited epidemiological research on repatriations is available , 10, 11,The cost of repatriation is an unexplored field and careful attention should be paid to the costing methodology as at the moment there is no golden standard. It is crucial that the cost formula represents the relevant costing categories, is reliable, valid, and user friendly in order to provide useful data and be implemented in the shipping industry and at the ship level .The European Union HealthBASKET project proposed a case vignette method to estimate the costs of treatment. The case vignette is an innovative and novel approach developed to explore resource use and costs.A case vignette describes a typical patient in regards to diagnosis, age, gender and possible comorbidity and is a retrospective episode-specific costing approach. The approach was applied to the vignette cases – diagnoses – in order to compare the costs of ten different treatments under DRG tariffs across nine European Union countries , 18.Even though research on the evaluation of the financial burden of a range of diseases in different countries and public administration levels is widely available, very limited research concerns the shipping sector.The objective of this study was to estimate the average total costs of repatriating seafarers based on four case vignettes.Four case vignettes were used Table  based onThe primary data source for cost information was the Danish Maritime Authority (DMA). DMA reimburses the ship-owners costs of repatriation and keeps records of the reimbursements made. However, DMA keeps paper archives of all the reimbursement cases, making it highly difficult and time consuming to identify the relevant cases to fit the case vignettes. The identification of the cases to fit the case vignettes were instead derived from Radio Medical Denmark records, which contains information on the date of the call, the expected diagnosis, personal identification number, gender of the seafarer and whether a helicopter emergency medical service or deviation was used to get the seafarer from the ship to shore. For each case vignette, one anonymized match was derived from the Radio Medical Denmark records in the period 2011 to 2013 The survey was approved by the responsible Authority, the Danish Data Protection Agency.This study estimates the average total costs of repatriation with an employer perspective based on the local approach  and follCTotal = CDirect + CIndirect.The following cost formula was applied to estimate the average total costs of repatriation capturing both the direct and indirect costs of repatriation: The direct costs of repatriation are related to evacuation via helicopter or ship deviation, further transportation costs such as ambulance and airplane to repatriation country, hospitalization, medication, rehabilitation and sickness benefits, expressed in the formula :\documenCIndirect = Cproduction loss + Crecruit + Covertime + Cinsurance premium + Cmanage.The indirect costs are expressed as: Where:Cproduction loss: the costs resulting from a slowdown in production.Crecruit: the costs of hiring an additional worker to replace to repatriated seafarer.Covertime: the costs related to paying overtime to fellow seafarers in order to avoid a slowdown in production.Cinsurance premium: the costs related to an increase in insurance premium, which occurs after having the costs related to repatriation reimbursed.Cmanage: the costs related to managing the repatriation case e.g. the captain of the vessel must spend time compiling the receipts and claiming reimbursement [ursement .This formula is flexible enough to accommodate the data from DMA and company level.A detailed description of the resource utilization of each of the four case vignettes were created and the resource utilization for each case vignette was measured using the local approach , 17. TheThe fourth and final step was to measure resource units and place a monetary value on the consumption, taken into consideration both direct and indirect costs.The data for estimating the direct costs were retrieved from DMA based on the claims filed by the shipping companies . These cLess information was available to estimate the indirect costs including productivity losses, management of the case and recruitment of a new seafarer. Assumptions were based on the available literature Table . The friThe average total costs of the four case vignettes varied between 23,792 euro for the least expensive – the malaria case vignette – up to 98,823 for the most expensive – the AMI case vignette. Table The AMI case vignette was comprised of a male seafarer who was evacuated by helicopter to the port of Bergen, Norway in 2011. The treatment was a percutaneous coronary intervention (PCI) and he was hospitalized for 12 days before returning to his home country, where he received further treatment and hospitalization. The seafarer received sickness benefits for the full period of 18 weeks. The total average cost amounted to 98,823 Euro. The main cost driver for this repatriation case was the treatment costs, which accounted for 44.7% of total costs equaling to 44,174 euro.The malignant hypertension case vignette was comprised of a male engineer who was evacuated to the port of Shanghai for medical examination due to high blood pressure, in 2013. The ship had to deviate for 6 h to get to the port of Shanghai. The seafarer was found not-fit-for-duty and transported to his country of origin. The data from DMA provided no information on the sickness duration and it was assumed that the seafarer was ill for the full 18 week period that DMA reimburses. The average total costs were estimated at 47,597 euro. The main cost driver was the deviation cost, which accounted for 13,200 euro (27.7%) .The appendicitis case vignette was comprised of the ships cook turning ill with severe abdominal pain in 2011. She was evacuated to the port of Malaga. The ship had to deviate for 12 h to get to the port of Malaga, where the seafarer was transported to the hospital for an appendectomy. As illustrated in Table The malaria case vignette comprised of a deck officer. The seafarer was evacuated to the port of Norfolk Virginia after a rapid malaria test shown a positive result and from the port to the hospital for further diagnosis and treatment. A boat service was utilized to get the seafarer from the vessel to shore. The patient was initiated on malaria treatment and discharged from the hospital the following day. The seafarer was found not-fit-for-duty and had to be repatriated back to his home country. The total average cost amounted to 23,792 euro, with the main single cost driver the cost of a new recruitment, which accounted for 49.7% of the average total costs of this repatriation case equivalent to 11,825 euro.A sensitivity analysis was carried out for all the four cases in order to investigate the robustness of the cost categories. The sensitivity analysis addressed the uncertainties regarding the cost estimates and assumptions . The senThe sensitivity analysis revealed that deviation and evacuation had major impact on the average total costs of repatriation. More specifically, in the appendicitis case the average total costs changed by as much as 23% by applying the pessimistic and optimistic costs for deviation. For the AMI case vignette the tornado diagram Fig.  illustraI) the costs of evacuation (getting the seafarer from the vessel to shore) and II) differences in treatment costs. The variations in indirect costs between the cases were mainly due to differences in the replacement costs. The average indirect costs varied between 14,000 euro in the malaria case and almost 22,000 euro in the malignant hypertension case vignette. The variation indicates that average indirect cost proportions ranges between 22 and 60% in the case vignettes. Indirect costs remain a substantial cost driver and outline the importance of estimating indirect costs, when estimating the total costs of repatriation.Limited published literature is available on the topic of repatriating seafarers , showingIn the AMI case vignette the data revealed, that the employer had costs which were not reimbursed by DMA and the estimate of 98,823 euro is, therefore, likely to be an underestimate of the financial burden held by the employer in this repatriation case. This illustrates that using the data from DMA as a proxy for the direct costs held by the employer is not entirely accurate.Previous data suggests that the annual number of repatriations amounts to nearly 10,000 with an annual cost of 760 million euro . With thResults from similar studies using the case vignette approach to estimate the costs of AMI treatment in nine selected countries under DRG tariffs showed big differences among countries too. In France, The Netherlands and Italy, PCI was the standard of care intervention to treat AMI, with costs ranging between 3720 and 9374 euros . In caseSimilar results were found with regards to the costs of treatment for appendicitis . The meaThis implies that in the case of seafarers – international employees not covered by any health system – market prices are used for the health services provided to them, contributing to a higher treatment cost.The cost of helicopter evacuation at sea was estimated to be 25,000 euros per intervention , 35.A considerable amount of indirect cost represented the ship deviations depending on the distance from the nearest shore.Any model is always a simplification of the reality , and sinThis study did not estimate any costs in terms of charterer loss penalties, which could imply that estimates of repatriation in the current study are conservative estimates of the true total costs.By applying the case vignette approach, it was assumed that cases in each case vignette represents the typical repatriation case of this disease, however a more representative sample could provide more accurate cost data.I) acute myocardial infarction, II) malignant hypertension, III) acute appendicitis and IV) malaria. The findings are a framework for investigating the average total costs of repatriation by applying a local level micro-costing approach. The cost formula included direct and indirect costs and the local approach proved to be a feasible approach to estimate the total costs of repatriation with an employer perspective.This study was a first attempt to map the relevant financial burden to the employer due to employee sickness on board merchant vessels. The objective was to estimate the costs of repatriation of ill seafarers based on four case vignettes; Every case of repatriation poses a large financial burden on employers, and the results indicated a large variation in the average total costs of repatriation for the four case vignettes. These variations in the proportions of direct cost were mainly due to hospitalization and deviation expenses. It is worth noticing that fee-for-service contributed to higher prices for treating seafarers around the globe. This clearly shows how prices for treating the same diagnosis differentiate in different countries and give insight for introducing possible collective bargaining agreements.With regards to the indirect costs the recruitment of a new employee was the main cost driver among the case vignettes. In our analysis, it was established that indirect costs are an important estimate from the employer’s point of view, and a major cost driver for the total costs of repatriation. These indirect costs are not reimbursed, and they all fall directly upon the employer. In order to estimate the total costs of repatriation, it would be beneficial if these cost data were collected regularly at the company level.From the employers’ point of view, it would be interesting to know these costs of repatriation, especially regarding helicopter evacuation, deviation and charterer loss. This could help the employers’ insight and motivation for disease preventive and health promotion interventions on board.Employers have a financial interest in promoting the health of seafarers by introducing or strengthening cost-effective prevention and health promotion programs, and hereby reducing the number of repatriations, as each repatriation of an ill seafarer is a heavy financial burden to the employer.


Foveavirus in the family Betaflexiviridae.Here, we report the genome sequence of grapevine virus T (GVT), a novel single-stranded RNA virus identified from a transcriptome of grapevine. The genome of GVT is 8,701 nucleotides in length and encodes five open reading frames. GVT is a putative member of the genus  Vitis vinifera) are commercially important fruit crops worldwide. Grapevines are cultivated for the production of wine or table grapes. A wide range of pathogens, including viruses, can threaten the quality and quantity of grapevine production. To date, more than 70 plant viruses infecting grapevines have been reported (Grapevines (reported .With the advancement of next-generation sequencing (NGS) techniques, numerous plant viruses, including novel viruses, have been identified , 3. NGS de novo assembled with Trinity version 2.0.6 with default parameters (https://www.ncbi.nlm.nih.gov/genome/viruses).In the search for viruses and viroids infecting grapevine using transcriptome data, a novel virus has been identified from a transcriptome of the grapevine cultivar Teroldego . Detailerameters . A BLASTFoveavirus in the family Betaflexiviridae. Taken together, we report a novel virus, GVT, which is a potential member of the genus Foveavirus, from the grapevine transcriptome.From the transcriptome of Teroldego, we identified 86 contigs associated with viruses and viroids. Of them, two contigs were associated with a novel virus named grapevine virus T (GVT) isolate Cho (GenBank accession number MF095096). The genome of GVT is composed of positive single-stranded RNA with a length of 8,701 nucleotides (nt). The obtained GVT genome sequence is a nearly full-length genome containing sequences of the 5′ and 3′ untranslated regions . The GVT encodes five open reading frames (ORFs), replicase (ORF1), three triple-gene blocks , and coat protein (ORF5). The BLASTp search against NCBI’s nonredundant protein database revealed that the five ORFs of GVT shared the following sequence similarities with grapevine rupestris stem pitting-associated virus (GRSPaV): 52% identity with replicase, 62% identity with TGB1, 53% identity with TGB2, 48% identity with TGB3, and 53% identity with coat protein. Based on the BLASTp results, we proposed that the identified GVT is a putative member of the genus MF095096.The full-genome sequence of grapevine virus T isolate Cho has been submitted to GenBank under the accession number


The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients (“high-risk combinations”) under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels. Historically, the impact of foreign animal diseases (FADs) on global livestock production and economics has been devastating . In 1997While the US has remained free of FMD and CSF over the past several decades, projected losses should an outbreak occur in the country range between $12.9-$14 billion for FMD and $2.6-$9.6 billion for CSF . In addiThe introduction of Porcine Epidemic Diarrhea Virus (PEDV) into the US in 2013 serves as an example of the impact that exotic diseases may have on the US livestock industry . AlthougThese results raised important questions as to whether contaminated animal feed and feed ingredients could serve as vehicles for the spread of other viral diseases between countries. Therefore, the objective of this study was to evaluate the survival of select viral pathogens in contaminated feed ingredients using models designed to simulate transportation conditions across different regions of the world. The study was based on the hypothesis that virus survival under the simulated transportation conditions would depend on the combination of virus and ingredient, with some ingredients providing the ideal matrix for survival of select viruses (high-risk combinations).http://www.swinehealth.org/swine-disease-matrix/), which consists of a list of important viral pathogens of swine that were ranked by SHIC based on three criteria: 1) likelihood of entry to the US or becoming an emerging disease if already endemic in the US, 2) economic impact on US production, and 3) impact on domestic and international markets. From this list, 11 pathogens were selected for this study: FMDV, CSFV, ASFV, IAV-S, PRV, NiV, PRRSV, SVDV, VSV, PCV2 and VESV.The Swine Health Information Center (SHIC) has recently compiled the swine disease matrix  laboratory conditions at South Dakota State University (SDSU), while the studies with ASFV were performed in BSL-3 laboratory conditions at the Biosecurity Research Institute (BRI) at Kansas State University (KSU). A complete list of target viruses, viral surrogates and their properties are presented in Since FMDV, CSFV, PRV, NiV, SVDV and VESV are exotic to the US and most are select agents, closely related surrogate viruses with similar genetic and physicochemical properties were selected . For FMDTo assess the survival of viral pathogens in feed ingredients that are imported into the US, two transboundary shipment/transportation models were developed: a Trans-Pacific model  and a TrThe website SeaRates.com was used to develop a representative route and timeframe that would model the shipment/transportation of feed ingredients from China to the US , 15. Thewww.wunderground.com), and the oceanic segment of the trip. The data used to generate the environmental curve represented a 37-day trip between Beijing and Des Moines during December 23, 2012 to January 27, 2013. To generate the 37-day environmental curve, available data summarizing temperature and % RH in shipping containers traveling from Asia to the US for the oceanic segments of the model  [To simulate environmental conditions ) that occur during shipment over land and sea, an environmental curve was generated based on historical temperature and % RH data available for both land segments   were paiwww.wunderground.com) for the land segments of the trip , to generate the 30-day environmental curve. To simulate the effect of daily environmental fluctuations, we used temperature and % RH data collected at four different times of the day . The environmental curve  and assumed that ASFV contamination of feed ingredients could occur in Warsaw, Poland, either at the manufacturing plant or post-processing, after the ingredients would travel across Western Europe, pass through Hannover, Germany to the port of Le Havre, France, where the cargo would be held for 7 days in preparation for shipment to the US. The cargo would then travel across the Atlantic Ocean over a 9-day period and enter the US at the port of New York City, NY. Following a seven day period to clear customs, it would then be transported for two days via Interstate 80, passing the cities of Cleveland, OH and Chicago, IL to the destination of Des Moines, IA, where it would remain for three days. In total, the transport period equaled 30 days. As with the Trans-Pacific model, an environmental curve consisting of historical temperature and % RH data was generated for both land segments (Europe and US) and the oceanic segment of the trip. The data used to generate the environmental curve represented a 30-day trip between Warsaw and Des Moines during April 5, 2011 to May 4, 2011. Historical temperature and % RH data from shipping containers travelling throughout the North Atlantic during the period of April 14–22, 2011  were comal curve  was progwww.hs.usitc.gov), which provides a transaction of specific trade commodities between the US and its international trading partners . Based on these transactions, a panel of 11 animal feed ingredients and or products of animal origin known to be imported into the US from Asia and/or Eastern Europe were selected. These included organic and conventional soybean meal, soy oil cake, dried distillers' grains with solubles (DDGS), lysine hydrochloride, vitamin D, choline chloride, moist cat food, moist dog food, dry dog food and natural pork sausage casings. The ingredients and products used in our study were obtained from US feed mills or stores, and the same batch of each ingredient was used for all samples across both models. Each ingredient and product were weighted into 5 g duplicate samples that were placed in 50 mL mini bioreactor tubes ; with vented caps to allow exchange of temperature and humidity between the interior and exterior of the tube. All samples were subjected to gamma irradiation  to eliminate microbial contaminants prior to use. Select ingredients were subjected to bromatological analysis. Ingredients were analyzed at Midwest Laboratories  and parameters evaluated included protein (crude), fat (crude), fiber (acid detergent), ash, moisture, dry matter, and pH.Different feed ingredients and/or products of animal origin were used to assess the survival of each target virus. These ingredients or products were selected based on the amount imported into the US . This inDuplicate samples of each ingredient were organized into four independent batches, with each batch representing a specific sampling point during the Trans-Pacific or Trans-Atlantic model . For the5 tissue culture infectious dose 50 (TCID50) of each virus [Five grams of gamma-irradiated ingredients (in 50 mL mini bioreactor tubes) were spiked with 100 μL of MEM  containing 1 x 10ch virus . Samples5 TCID50 of each respective virus and used as positive controls. Additionally, stock virus in MEM or RPMI  was added at the same concentration to an empty 50 mL mini bioreactor tube. The purpose of this control was to determine whether viruses could survive the simulated journey in the absence of a feed matrix. All controls were run in duplicate and were included in all sampling batches.Five grams of complete swine feed  were inoculated with PBS and used as negative controls in each sampling batch. Five grams of complete swine feed were spiked with 1 x 10All samples inoculated with the target viruses were incubated in environmental chambers ; Model 3911, Thermo Scientific Forma, Waltham, MA (Trans-Atlantic)) programed to simulate the environmental conditions (temperature and % RH) described above (Trans-Pacific and Trans-Atlantic models). Samples from Batches 1, 2, 3 and 4 were removed from the environmental chamber on appropriate sampling points (described above) and processed for testing by real-time PCR, virus isolation and/or swine bioassays. Each sample was re-suspended in 15 mL sterile PBS, vortexed for 10 seconds and centrifuged. The supernatant was aliquoted and centrifuged for 10,000g for 10 minutes prior to real-time PCR (PCR) and virus isolation. An additional aliquot was stored at -80°C for swine bioassay.All samples from Batches 1 and 4 were tested by real time PCR at the Animal Disease Research and Diagnostic Laboratory (ADRDL) at South Dakota State University or at the Biosecurity Research Institute at Kansas State University in the case of ASFV. These batches were selected to validate successful inoculation and determine the presence of viral nucleic acid at the beginning (Batch 1) and end (Batch 4) of the incubation period. A cycle threshold of ≥40 was considered negative for ASFV and a cycle threshold of ≥38 was considered negative for the remaining viruses.® CRL-5803™) while the CDV strain Snyder Hill (ATCC® VR-1587™) and the VSV strain , were grown in Vero cells (ATCC® CRL-1586™). MARC145 cells were used to amplify PRRSV strain SD-174. Feline Calicivirus strain FCV-2280 (ATCC® CCL-94™) was amplified in CRFK cells (ATCC® VR-2057™). The Swine Influenza virus H1N1 was amplified in MDCK.2 (ATCC® CRL-2936™), while Porcine Sapelovirus strain PS 32 (NVSL 059-PDV) was amplified in ST cells (ATCC® CRL-1746™) and the PCV2 , was amplified in PK15 cells free of PCV1. A splenic homogenate containing ASFV (Georgia 2007/1) was quantified on porcine alveolar macrophages (PAMs). The ASFV Georgia 2007/1 isolate was kindly provided by Linda Dixon at the Pirbright Institute and obtained through the generosity of David Williams at the Commonwealth Scientific and Industrial Research Organization’s Australian Animal Health Laboratory. With exception of H1299 cells (ATCC® CRL-5803™) and PAMs that were cultured using RPMI medium , the cells used for virus amplification, titration and isolation were cultured in minimal essential medium (MEM Corning). Both MEM and RPMI used to culture cells for virus amplification were supplemented with 2 mM of L-glutamine (Corning), 100 U/ml of penicillin (Gibco), 100 μg/mL of streptomycin (Gibco), 50 μg/mL gentamicin sulfate (VWR), 2.5 μg/mL of amphotericin B (Corning), and 5–10% of fetal bovine serum (Hyclone). MDCK cells used for IAV-S amplification and VI were maintained in MEM supplemented with 8% FBS, 5% 5 g/L lactalbumin enzymatic hydrosylate  and containing 100 U/mL of penicillin (Gibco), 100ug/L of streptomycin (Gibco), 50 ug/mL gentamycin sulfate (VWR) and 2.5 ug/mL of amphotericin B. Replacement media for IAV-S isolation consisted of MEM containing 5% LAH (5g/L) with penicillin/streptomycin, gentamycin and amphotericin B, supplemented with 10 ug/mL trypsin. All cells and fetal bovine serum tested negative for Bovine Viral Diarrhea Virus. For virus propagation, 75-cm2 flasks containing 70 to 85% confluent cell monolayers were inoculated with the appropriate virus and incubated at 37°C for 48 to 120 h depending on the virus strain. Following one freeze-thaw cycle, the suspension was centrifuged for 10 min at 1,000 × g. Cell supernatant was collected, aliquot, and stored at −80°C until use. The virus stocks were titrated in 96-well microtiter plates by endpoint dilution. Titers were calculated and expressed as median TCID50 [http://www.oie.int/manual-of-diagnostic-tests-and-vaccines-for-terrestrial-animals/). Finally, virus titrations were conducted in 96-well plates to determine the amount of viable virus (endpoint titers) present in duplicate samples at the end of the experiment (Batch 4).Appropriate cell cultures susceptible to each of the viruses in our study were used to amplify and to assess virus survival in feed samples collected on Batches 1, 2, 3 and 4 by virus isolation. Primary bovine turbinate (BT) cells were used to amplify BVDV-1a strain Singer (NVSL-140-BVDV) and BHV-1 isolate SD1331. SVA strain SD15-26 [n TCID50 . Virus iAll procedures involving animals were reviewed and approved by the SDSU or KSU Institutional Animal Care and Use Committees . All animals were monitored 2–3 times a day by experienced animal care takers under the supervision of SDSU’s and KSU’s veterinarians. Parameters monitored included characteristic clinical signs for each of the target pathogens . Each animal had its own medical record and supportive treatment consisting of electrolyte solutions and/or anti-inflammatories were administered at the discretion of the attending veterinarians. Animals that were severely sick and were unable to stand/walk or eat and drink were sedated and humanely euthanized . The swine bioassay was used to determine whether viable virus was present in feed ingredient samples that tested positive on PCR but were negative on VI in cell culture. As a porcine model was used, bioassays were performed only on samples inoculated with swine pathogens including SVA, PRRSV, PCV2, PSV, IAV-S and ASFV. Bioassays were conducted under Biosafety Level 2+ conditions at the Animal Resource Wing (ARW) at SDSU  or underth side consisting of bars and a gate. The room was environmentally controlled and complete exchange of air within the room occurred 14.5 times/hour. To reduce the risk of aerosols in the room, the pens were not sprayed with water during the 5-day post-challenge period.The ASFV bioassay was conducted with weaned pigs (approximately 21-days old), obtained from a high health commercial source. All pigs were housed in a 66 square meter room and maintained under BSL-3 Ag containment conditions. Pigs were individually housed in 1.8 square meter pens with each pen separated by approximately 2.3 meters. Pens were raised, stainless steel decks with slotted fiberglass flooring. Pens had 3 solid sides with the 4The inocula used for the swine bioassays were prepared as described above. Each piglet was inoculated with 1 mL of the cleared supernatant from processed feed ingredients and monitored for 5–7 days. Piglets inoculated with PRRSV, PCV2 and ASFV were injected via the intramuscular (IM) route, those inoculated with IAV-S were inoculated via the intranasal route (IN), while piglets inoculated with PSV and SVA received the inoculum orally (PO). A negative control piglet  was included in all bioassays.Following inoculation, the health status of each piglet was monitored daily. Blood samples were collected from piglets inoculated with samples spiked with PCV, PRRSV, SVA and ASFV, nasal swabs were collected from piglets inoculated with samples spiked with IAV-S and rectal swabs were collected from piglets inoculated with samples spiked with PSV. Samples were submitted to the SDSU ADRDL and tested by PCR for the specific viruses. Samples from the ASFV bioassay were tested at the KSU BRI. If infection was diagnosed in an animal or a specific unit, all animals were sampled and humanely euthanized.50), half-life (T ½) estimates were calculated. The T ½ analysis was performed by fitting a linear regression model to the data using the SAS 9.4 Proc Reg procedure for each ingredient and virus, using the log of the viral concentration (inoculation dose and endpoint titer) as the response variables and time  as the explanatory variables. The slope of the respective lines were calculated from these models and half-lives was estimated using -log (2)/slope estimates as previously described [Based on initial inoculation dose and Batch 4 endpoint titers , lysine hydrochloride and complete feed (n = 5), vitamin D, choline chloride, and sausage casings (n = 4), organic soybean meal, the three types of pet food (n = 3) and DDGS (n = 2). To determine whether specific characteristics of those ingredients could be associated with virus survival, a complete bromatological analysis was conducted on samples of choline chloride, vitamin D, lysine hydrochloride, soy oil cake, DDGS, dry dog food and conventional and organic soybean meal  did not survive in the absence of a feed matrix (stock virus controls). These stock virus controls were included in the experimental design to assess whether a virus could survive under the simulated environmental conditions used in the study in the absence of a feed matrix. It is important to note that both models involved moderate temperatures (4–14°C during the Trans-Pacific model and 10–20°C during the Trans-Atlantic model) and broad relative humidity levels (25–95% RH Trans-Pacific and 40–90% RH Trans-Atlantic) which fluctuated daily. The fact that 11 of 12 viruses did not survive these conditions when deposited directly into polypropylene tubes but survived in the presence of feed ingredients suggest that the feed matrix may have provided protection from the environmental conditions encountered during the simulated transport.Upon careful critique of our study, it is evident that the experimental design presented several strengths. It used rigorous transport models, capable of simulating both Trans-Pacific and Trans-Atlantic shipments of cargo, representative timelines, shipping routes, environmental conditions and ingredients, along with samples that were repeatedly tested using complementary assays. While originally designed to use surrogate viruses, our study assessed the survival of the actual target pathogen in 6 of 12 cases, enhancing the significance of the results. This is evidenced with results demonstrating the survival of ASFV, one of the most significant threats to the swine industry worldwide. While it was not possible to work with pathogens such as FMDV or SVDV, we selected the surrogates SVA and PSV, which are important pathogens in their own right that are closely related to the target pathogens by viral family, presenting similar structure, morphological features and physicochemical properties. In addition, the calculation of half-life was important for several reasons, as it provided a means to compare viral survival across feed ingredients, is independent of viral inoculation dose, and may help assist with biosecurity decision-making, specifically the proper storage time necessary to minimize viral survival in imported ingredients. Finally, the inclusion of multiple controls, including complete feed positive controls, complete feed negative controls and stock virus controls as described earlier, also strengthen our experimental design.The limitations of the study also need to be discussed. First, it is important to mention that the study used models performed under controlled laboratory conditions which required the use of surrogate viruses in certain cases; therefore, we do not know if FMDV, CSFV, or PRV, for example, would survive the journey, if subjected to these transportation models. Because it was a model, the study was performed in small scale, using volumes of feed that were manageable under laboratory conditions and certainly do not represent real world situations. In addition, the sample sizes used across ingredients tested was small, resulting in insufficient data points to calculate confidence intervals around the half-life estimates. Furthermore, all samples were spiked with an equal amount of virus, which may or may not reflect the actual level of contamination that would occur in the field. As this information is currently unavailable for all target pathogens, we relied on data from viral load in feed from field cases of PEDV . FurtherIn conclusion, this study has provided new information supporting the hypothesis that the “high-risk combinations” i.e. “the right virus paired with the right ingredient”, may be a mechanism for the transboundary transport of pathogens, which is clearly a new paradigm. These data are applicable to viral diseases across many livestock species, including several diseases of swine, ruminants , avian (IAV-S) and humans . In addition, development of the transboundary model provides a platform by which further testing can be performed using other infectious or toxic agents, such as bacteria or chemicals, which will help to build a database regarding the risk of introducing contaminants through feed. In the end, it is hoped that the results of this study will stimulate communication and collaboration between the feed and livestock industries, resulting in further research into the emerging concept of “global feed biosecurity”. Ideally, this new information will enhance the accuracy of risk assessments, promote the development of efficacious feed-based mitigation strategies, and ultimately result in a change in philosophy regarding the global trade of feed ingredients from one that is based on price, to one where country of origin health status is a major consideration.


Plants respond to pathogens using an innate immune system that is broadly divided into PTI (pathogen-associated molecular pattern- or PAMP-triggered immunity) and ETI (effector-triggered immunity). PTI is activated upon perception of PAMPs, conserved motifs derived from pathogens, by surface membrane-anchored pattern recognition receptors (PRRs). To overcome this first line of defense, pathogens release into plant cells effectors that inhibit PTI and activate effector-triggered susceptibility (ETS). Counteracting this virulence strategy, plant cells synthesize intracellular resistance (R) proteins, which specifically recognize pathogen effectors or avirulence (Avr) factors and activate ETI. These coevolving pathogen virulence strategies and plant resistance mechanisms illustrate evolutionary arms race between pathogen and host, which is integrated into the zigzag model of plant innate immunity. Although antiviral immune concepts have been initially excluded from the zigzag model, recent studies have provided several lines of evidence substantiating the notion that plants deploy the innate immune system to fight viruses in a manner similar to that used for non-viral pathogens. First, most R proteins against viruses so far characterized share structural similarity with antibacterial and antifungal R gene products and elicit typical ETI-based immune responses. Second, virus-derived PAMPs may activate PTI-like responses through immune co-receptors of plant PTI. Finally, and even more compelling, a viral Avr factor that triggers ETI in resistant genotypes has recently been shown to act as a suppressor of PTI, integrating plant viruses into the co-evolutionary model of host-pathogen interactions, the zigzag model. In this review, we summarize these important progresses, focusing on the potential significance of antiviral immune receptors and co-receptors in plant antiviral innate immunity. In light of the innate immune system, we also discuss a newly uncovered layer of antiviral defense that is specific to plant DNA viruses and relies on transmembrane receptor-mediated translational suppression for defense. Figure 1). PRRs recognize conserved structural motifs present in microbes, which are known as microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs), or endogenous danger signals released by the plant during wounding or pathogenic attack, which are termed damage-associated molecular patterns . These intracellular receptors directly or indirectly recognize effectors secreted by pathogens into the host intracellular environment and activate effector-triggered immunity . Activation of NIK1-mediated antiviral signaling leads to translocation of the ribosomal protein L10 (RPL10) to the nucleus, where it interacts with L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB) to fully repress expression of translational machinery-related genes and global host translation. Begomovirus mRNAs are unable to escape this translational regulatory mechanism of plant cells and hence are not efficiently translated, which compromises infection upon activation of NIK1-mediated defense. Although the NIK1-mediated defense response is remarkably dissimilar from the PTI response, structural components and activation of the NIK1 immune receptor as well as its interaction with virus infection exhibit features reminiscent of the plant innate immunity mechanism.An additional recently uncovered virus-specific defense mechanism relies on suppression of host translation mediated by the transmembrane immune receptor NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1 (NIK1), which was first identified as a virulence target of begomovirus NSP , Tomato bushy stunt virus (TBSV), Cauliflower mosaic virus (CaMV) and Tomato mosaic virus  product, can lead to the hypersensitive reaction (HR). HR is considered a resistance response against several different pathogens that, to the some extent, occurs through similar mechanisms. Similar to non-viral infections, the HR response during viral infection is initiated by direct or indirect Avr-R interactions and is frequently associated with accumulation of SA in both infected and non-infected tissues . HR is auring HR . Althougus ToMV; , 2009. Fus ToMV; , 2009.Table 1). Based on their variable N-terminal domain, these plant NB-LRR proteins are further classified into coiled-coil (CC)-NB-LRR or Toll/interleukin 1 receptor-like (TIR)-NB-LRR protein families ] proteins that mediate resistance via specific (direct or indirect) recognition of a virus Avr factor  (Table 1families . Most offamilies . Recognifamilies  or throufamilies . In the families . Alternafamilies . TherefoTable 1)   . AnotherTable 1) , PHYTOALTable 1) , 2005 anTable 1) . The HRTTable 1) . DisruptTable 1)  and EDS1Table 1)  protein N gene (for necrotic-type response), which confers resistance against TMV and encodes a TIR-NB-LRR protein, was the first identified R gene . One such example is the tomato Tm-1 gene, which confers dominant resistance to ToMV and contains two conserved domains: an uncharacterized N-terminal region (residues M1–K431) and a TIM-barrel-like C-terminal domain . The Ty-1 and Ty-3 genes are allelic and code for an RDR of the RDRc type, which has an atypical DFDGD motif in the catalytic domain (Ty-1/Ty-3 plants display enhanced siRNA levels that coincide with hypermethylation of the TYLCV V1 (CP) promoter, indicating that Ty-1-based resistance against TYLCV involves enhanced transcriptional gene silencing.A few dominant resistance genes encoding the non-NB-LRR class of proteins have been characterized; these proteins have been found to function as sensors of virus infection but do not induce typical ETI-like defense responses, such as HR (c domain . The mecc domain . AccordiTable 1), which specifically recognize viral effectors or Avr factors and utilize signaling modules such as SGT1/RAR1/HSP90 and EDS1/PAD4/SAG101 complexes to mediate resistance responses, similar to non-viral pathogens. Therefore, plants appear to have evolved strategies and signaling modules to defend themselves against a large spectrum of pathogen types, such as bacteria, viruses and fungi. This interpretation allows us to integrate some aspects of the antiviral immune concepts into the typical bacterial and fungal immunity models to classify viral effectors and viral ETI.In summary, most antiviral dominant resistance genes so far characterized encode typical NB-LRR R proteins , have important roles in antiviral defense via recognition of a different range of MAMPs, such viral RNA and DNA . In contnfection . Consistt manner . The Ararus PPV; . Furtherus BPMV; , and chius BPMV; .Oilseed rape mosaic virus (ORMV) showed significantly reduced viral accumulation in treated leaves. Interestingly, dsRNA-mediated PTI appears to be independent of the RNA silencing pathway but does involve the co-receptor kinase SERK1. These findings relate membrane-associated signaling events with dsRNA-mediated PTI in plants  and virus-derived dsRNA have been shown to function as viral PAMPs in Arabidopsis and to induce the PTI pathway . Indeed,n plants . Althougin planta during infection (Table 1). These observations suggest that plant viruses also fit into the zigzag model of co-evolving pathogenic virulence strategies and plant defense responses that shape the two-branched innate immune system .The transmembrane receptor NIK was first identified as a virulence target of Nuclear Shuttle Protein (NSP) from e family . Similare family . NeverthNUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1-mediated antiviral signaling is activated upon perception of begomovirus infection, which leads to phosphorylation of the NIK1 kinase at key threonine residues at positions 468 and 474 . Thr-468In general, ligand-dependent phosphorylation and activation of RLKs require homo or heterodimerization of the receptors. In the case of BAK1 and SERK1, compelling evidence has revealed that they function primarily as co-receptors for receptor signaling not only in defense but also in development . As a mein vitro and in vivo with the kinase domain of NIK1 to suppress NIK1 activity (Bean dwarf mosaic virus (BDMV) has been demonstrated to function as an Avr gene and elicit HR in Phaseolus vulgaris  infection . Activated NIK1 mediates phosphorylation and consequent translocation of RPL10 to the nucleus, where it interacts with LIMYB to fully repress RP gene expression. Prolonged down-regulation of RP gene expression leads to suppression of global host translation. DNA viruses, such as begomoviruses, cannot escape this translational regulatory mechanism of plant cells, and viral mRNAs are not translated efficiently, thereby compromising infection. NSP acts as a virulence factor and suppresses the kinase activity of NIK1 to overcome the NIK1-mediated immune response. NSP from the begomovirus BDMV has also been shown to function as an Avr factor that activates typical ETI responses in resistant bean genotypes. Therefore, NSP may link the suppression of NIK1 signaling with activation of ETI responses in accordance with the zigzag evolutionary model of plant innate immunity, although the NIK1-mediated antiviral signaling may represent a new evolved branch of plant antiviral immunity, which relies on suppression of translation for defense.Although NIK1 is structurally similar to SERKs, the mechanism of NIK1-mediated antiviral defense is distinct from that of BAK1-mediated PTI. The current model for NIK1-mediated antiviral signaling states that, in response to virus infection, NIK1 undergoes homo- or heterodimerization to promote phosphorylation of the activation loop  and NB-LRR host targets (R proteins) and their mode of action in activating ETI and/or suppressing PTI will help to define the evolutionary pressure acting upon the host and viruses and to determine how to deploy the immune system for more efficient control of virus infection. We also need to define NIK1-mediated suppression of translation as a general or virus-specific antiviral strategy in plants. To date, a sustained NIK1 pathway has been shown to be effective against begomoviruses, one of the largest groups of plant DNA viruses, which cannot circumvent the regulatory mechanism of host translation. In this regard, the intrinsic capacity of agronomically relevant crops to withstand the deleterious effects of suppression of global translation must be considered as a relevant agronomic trait if we are to use the translational control branch of NIK1-mediated antiviral signaling for crop protection against begomoviruses.BG and IC wrote the first draft of the manuscript; JM wrote the ETI section; AS edited the manuscript and EF edited 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.


Aedes aegypti mosquitoes. Our findings suggest that Zika virus in Cambodia belongs to the Asia genotype, is endemic, has low prevalence, and has had low-level impact on public health.We describe a retrospective study on circulation of Zika virus in Cambodia during 2007–2016 among patients with dengue-like symptoms and  Flaviviridae, genus Flavivirus) is an arthropodborne virus mainly transmitted by the Aedes mosquito. Zika virus was first isolated in 1947 in Africa from rhesus macaques, and human illness was first recognized in Uganda in 1964 . Acute- and convalescent-phase samples from ≈5 patients randomly selected from each of the 5 sentinel sites in the NDCP system are collected and tested each week at Institut Pasteur du Cambodge (IPC) to ensure virologic diagnosis and surveillance . In total, 2,400 serum samples and 173 supernatants from C6/36 cell cultures of serum from the dengue surveillance system, 270 urine samples from the DENFREE study, and 3,159 mosquitoes were tested for Zika virus by qRT-PCR (We conducted a retrospective study by using samples in our biobank: 1) acute-phase serum and supernatant from NDCP collected during January 2007–July 2016; 2) acute- or convalescent-phase serum and supernatant samples collected by private clinics for dengue diagnosis during the same period; and 3) urine from dengue-negative patients and mosquito samples included in the DENFREE study conducted in Cambodia during 2012–2013 . The useFive human serum samples were positive for Zika virus by qRT-PCR; these samples were collected in 2007 (n = 1), 2008 (n = 1), 2009 (n = 2), and 2015 (n = 1) . No othe>2 of 3 antigens tested ); and 1,087 samples negative for antibodies for any of the 3 viruses tested.The serologic study of 1,992 acute-phase serum samples showed that 16 samples from 2007 (n = 2), 2008 (n = 1), 2010 (n = 2), 2012 (n = 4), and 2015 (n = 7) were positive for Zika virus IgM, with no cross-reactivity with DENV and JEV, suggesting a presumptive diagnosis of recent infection. The interpretation of the 1,976 remaining samples is distributed as follows: 146 recent DENV infections; 4 recent JEV infections; 743 recent flavivirus infections  the low incidence of Zika virus, which usually causes mild disease compared with DENV, in Cambodia; 2) possible bias in the selection of viremic cases, which were mainly reported in hospitalized suspected dengue patients and therefore were more severe; 3) less than ideal timing of specimen collection for the detection of Zika virus, which has been shown to be transient and frequently low-titer and too early for detection of Zika virus IgM on acute-phase serum samples; or 4) the difficulty in interpreting MAC-ELISA results owing to the cross-reactivity of IgM between DENV and JEV, which are widely present in Cambodia.Ae. aegypti and Ae. albopictus mosquitoes are widespread throughout Cambodia. The risk for reemergence or reintroduction of Zika virus should be monitored by extending the existing surveillance program to ambulatory suspected dengue patients and should be completed by a focal entomological study. Moreover, in the absence of a previously detected Zika outbreak in Cambodia, seroprevalence studies in the general population should help estimate the size of the population remaining susceptible to Zika infection.The epidemiology of Zika virus is changing, and the risk for reintroduction in Cambodia is a real threat that should not be underestimated. Thailand, Vietnam, and Laos have reported cases since December 2015, and, local transmission has been observed recently in Singapore (Description of the DENFREE study conducted in Cambodia during 2012–2013.Samples collected from 5 persons in Cambodia in 2007, 2008, 2009, and 2015 that tested positive for Zika virus by quantitative RT-PCR.