Culex spp. mosquitoes and water birds across a large swath of southern Asia and northern Australia. Currently JEV is the leading cause of vaccine-preventable encephalitis in humans in Asia. Five genotypes of JEV (G-I–G-V) have been responsible for historical and current outbreaks in endemic regions, and G-I and G-III co-circulate throughout Southern Asia. While G-III has historically been the dominant genotype worldwide, G-I has gradually but steadily displaced G-III. The objective of this study was to better understand the phenomenon of genotype displacement for JEV by evaluating both avian host and mosquito vector susceptibilities to infection with representatives from both G-I and G-III. Since ducks and Culex quinquefasciatus mosquitoes are prevalent avian hosts and vectors perpetuating JEV transmission in JE endemic areas, experimental evaluation of virus replication in these species was considered to approximate the natural conditions necessary for studying the role of host, vectors and viral fitness in the JEV genotype displacement context. We evaluated viremia in ducklings infected with G-I and G-III, and did not detect differences in magnitude or duration of viremia. Testing the same viruses in mosquitoes revealed that the rates of infection, dissemination and transmission were higher in virus strains belonging to G-I than G-III, and that the extrinsic incubation period was shorter for the G-I strains. These data suggest that the characteristics of JEV infection of mosquitoes but not of ducklings, may have play a role in genotype displacement.Japanese encephalitis virus (JEV) is a flavivirus that is maintained via transmission between  Flaviviridae and genus Flavivirus, and is an enveloped, single-stranded positive-sense RNA virus having a genome approximately 11 kb in size [Culex tritaeniorhynchus, transmits the virus via bite to ardeid birds such as herons and egrets (reservoir hosts) and pigs (amplifying host) [Japanese encephalitis virus (JEV) belongs to the family  in size . JEV exing host) ,4. The mng host) ,4,5. Thing host) ,7. JEV ing host) ,8. With ng host) ,8,9,10. ng host) ,11,12.Japanese encephalitis virus is thought to have emerged from ancestral viruses during the 19th century in the region of the Malay . HistoriAedes albopictus C6/36 cells and duck embryo fibroblasts) approaches to explain the JEV genotype displacement and found that JEV G-I isolates indicated significantly higher infectivity titer than JEV G-III isolates at 24, 36, and 48-hour post-infection in C6/36 cells. Another study tested susceptibilities of Cx. quinquefasciatus to JEV G-I and G-III strains, and found North American Cx. quinquefasciatus as a competent vector for JEV G-I and G-III strains but JEV G-III demonstrated significantly higher infection rate than JEV G-I and non-demonstrable difference in transmission rate between both genotypes [The mechanisms responsible for dominance of G-I strains and displacement of G-III strains remains poorly understood. Studies based on nucleotide sequence analysis have described genetic diversity, evolutionary history, and host composition over the course of genotype displacement ,26,37. Senotypes . Howeverenotypes  incorporThe study was approved by the Animal Care and Use Committee of Colorado State University (protocol number: 16-6477A) and was conducted in biosafety level-3 facility approved by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC).2. Stock viruses were inoculated in Vero cells and fresh viruses were harvested at the end of 4 days post-inoculation (dpi). The strains of JEV utilized in this study are described in Vero  cells, a mammalian cell type that does not possess functional interferon signaling, obtained from the American Type Culture Collection  were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% bovine calf serum (BCS), penicillin (100 U/mL) and streptomycin (50 μg/mL) at 37 °C with 5% CO2, virus inoculum was removed, cells were washed once with PBS and 5 mL fresh growth media (DMEM with 5% FBS) was added. Samples for virus titration were collected on the day of inoculation , and daily for 8 days; at each sampling, 0.5 mL of supernatant was harvested and replaced with the same amount of fresh growth media. The supernatants were supplemented with 0.1 mL FBS and stored at −80 °C until assayed by plaque assay in Vero cells.Five replicates of a multi-step growth curve in Vero cells were obtained for all six viruses using a multiplicity of infection (MOI) of 0.01. Briefly, after inoculating the cells with viruses, the plates were incubated for 2 h at 37 °C with 5% COAnas platyrhynchos domesticus obtained from Murray McMurray Hatchery, Webster City, IA, USA) were housed under biosafety level-3 containment. Thirty ducklings were randomly allocated to six groups, corresponding to the six virus strains to be tested. Ducklings were banded with their specific numbers assigned to them. One extra duckling was added to either groups inoculated with MAR864 and Sagiyama strains, but was not inoculated and served as contact control. The duckling-cages were kept away from each other to prevent any contacts between groups. The ducklings were fed standard waterfowl feed and had water available ad libitum. The ducklings were inoculated subcutaneously with ~106 PFU of each virus at 5 to 6 days of age. A 100 μL blood sample was withdrawn from each duckling daily and immediately diluted into 450 μL of BA-1 medium , penicillin  and streptomycin (50 mg/L)) yielding a serum dilution of approximately 1:10. Body temperature and weight were measured three times at a single time-point and average measurement was recorded daily from the day before inoculation through 7 dpi and on 10, 14, and 21 dpi. Oral and cloacal swab samples were taken on 3, 4 and 5 dpi and immediately added to 450 μL of BA-1 medium supplemented with amphotericin B (2.5 mg/L), polymyxin B . and twice the standard concentration of penicillin, streptomycin, gentamicin. The samples were immediately stored at −80 °C until assay. All ducklings were euthanized on 21 dpi.Indian runner ducks , and when pupae started emerging, they were transferred in a cup with water inside a container covered with a mesh. The insectary was maintained at 26–27 °C, 16:8 light: dark cycle, and 70–80% relative humidity. Female mosquitoes of the F11–F13 generations (5–7 days post-eclosion) were used in this study.2 flasks were inoculated with each of the six strains of JEV G–I and G-III at an MOI of 0.01. Four days later, the supernatant from each flask was collected, centrifuged  to remove cellular debris, and the fresh, clarified supernatants were used for feeding mosquitoes. Mosquitoes were fed with defibrinated cattle blood mixed with freshly harvested viruses at a 1:1 ratio that was titrated at the time of use by plaque assay in Vero cells. The blood-virus mixture was pipetted into the loading chamber of a Hemotek membrane feeding system  covered with pork casing , and the mosquitoes were allowed to feed for 1 h at ambient room condition at 26 °C and 80% relative humidity. Blood-engorged mosquitoes for each virus strain were briefly cold anesthetized (4 °C) and separated into two groups for assay at day 7 and 14 post-feeding (dpf). The blood-engorged mosquitoes were incubated at 26 °C and 70% relative humidity with ad libitum water and sugar cubes. On 7 and 14 dpf, 60 mosquitoes per virus strain were cold anesthetized (4 °C), and legs and wings were removed and transferred into a clean tube containing two stainless steel beads and 250 μL mosquito diluent ). The mosquito bodies were placed with their proboscis inserted in a capillary tube containing immersion oil (about 5 μL) for at least 30 min. After salivation, mosquito bodies were transferred into a separate tube with mosquito diluent and beads. The ends of capillary tubes containing immersion oil and saliva were broken off into micro centrifuge tubes containing 100 μL mosquito diluent. Mosquito tissues  were homogenized at 25 cycles/second for 1 min in a mixer mill and subsequently centrifuged at 15,000× g for 3 min at 4 °C. To determine the infection status of mosquito tissues and saliva, 50 μL of each sample was inoculated onto Vero cell monolayers in two replicate wells of a 96-well plate and incubated at 37 °C. After incubation for an hour, 150 μL of DMEM supplemented with 10% FBS was added into each well and the plates were incubated for 24 h at 37 °C. Media from the 96-well plates inoculated with mosquito samples was decanted at 24 h; cells were washed once with PBS, fixed with 70% acetone for 1 h and dried. The cells were stained by addition of 50 μL of diluted mouse anti-JEV hyper-immune ascitic fluid  (1:100 in PBS) for 1 h at 37 °C, washed in PBS, then incubated for 1 h with 50 μL of DyLight conjugate of Goat anti-Mouse Ig  . The plates were washed again with PBS and observed under a fluorescent microscope. The sample was determined positive if replicate wells demonstrated fluorescence and negative if none of the wells demonstrate fluorescence. Controls in each assay consisted of duplicate wells inoculated with BA-1 diluent or BA-1 spiked with stock JEV. The infection rate was determined as the proportion of mosquito bodies that were found infected to the total number of mosquitoes exposed to the infectious blood meal. Dissemination rate was determined as the proportion of mosquito legs and wings that were found infected to the total number of mosquitoes exposed to the infectious blood meal. Similarly, transmission rate was determined as the proportion of mosquito saliva samples that were found infected to the total number of mosquitos exposed to the infectious blood meal.Vero cells cultured in 25 cm90).Replication of JEV in ducklings was measured as viremia titer. Plaques were counted on one and two days after the second overlay, and viremia titers were expressed as plaque forming units (PFU)/mL. The minimum concentration of JEV in serum or swab samples that could be detected using this assay was set at ~100 PFU/mL. Neutralizing antibody titers in sera were determined by plaque reduction neutralization test (PRNT). Briefly, 2-fold serial dilutions (starting at 1:10) of heat-inactivated (56 °C for 30 min) duckling sera were mixed with equal volumes of stock virus  diluted to ~200 PFU/mL. After 1 h of incubation, the serum-virus mixtures were inoculated into wells in 6-well plates of confluent Vero cells and processed subsequently as described for plaque assay. Plaques were counted one day after the second overlay and neutralization titer was expressed as the reciprocal of the highest dilution of the serum that inhibited ≥90% of JEV plaques (PRNTp < 0.05.Virus titer in Vero cells culture and viremia titer in ducklings were expressed as mean (±1 SE). The mean peak virus titer of each of the six JEV strains inoculated in Vero cells were compared using one-way ANOVA, and the time x strain interaction was analyzed by repeated measures ANOVA. Similarly, the mean peak viremia titers of the six strains in ducklings were compared using one-way ANOVA. Furthermore, infection and dissemination percentages were arcsine-square root transformed to render them normal, normality was checked on transformed data and compared by standard least squares method between G-I and G-III in the mosquito study. Since the data on transmission proportions cannot be normally distributed, as 50% of the values are tied 0’s, transmission proportions for G-I on 7 dpf, G-III on 7 dpf, G-I on 14 dpf and G-III on 14 dpf were pooled and compared by Fisher’s exact test. SAS 9.4  was used for all statistical analysis. Graphs were made on GraphPad Prism 7.0 . The statistical significance was declared at 10PFU/mL): KE-093-83 (8.6 ± 0.3), MAR864 (8.5 ± 0.2), JE-91 (8.8 ± 0.1), CH392 (9.0 ± 0.04), and Sagiyama (8.7 ± 0.2). The JKT27-087 virus achieved peak titer on 3 dpi (8.1 ± 0.3). When comparing only the mean peak virus titer (2 dpi), one-way ANOVA revealed a significant difference in the peak virus titer among strains , and Tukey-Kramer post-hoc analysis indicated that the mean peak virus titer of JKT27-087 differed significantly from the mean peak virus titers of the rest of the strains (p < 0.05). Similarly, the mean peak virus titers of G-I (8.6 ± 0.1) and G-III (8.4 ± 0.2) were not significantly different . Repeated measures ANOVA revealed a significant effect of strain on virus titer  and a significant time x strain interaction among six strains , and post-hoc Tukey-Kramer multiple comparison revealed only CH392 strain significantly different than JKT27-087 strain (p = 0.009). Virus titer for all strains declined after 3 dpi, corresponding to the observation of cytopathic effect in the Vero cells.The growth kinetics of all six strains of JEV were comparable in Vero cells . Five ofNone of the ducklings inoculated with JEV showed apparent signs of disease or distress. A mild elevation of body temperature (preinoculation body temperature range was 40.0–42.8 °C) on 1 dpi was observed in ducklings inoculated with MAR864, CH392, JKT27-087, and Sagiyama strains, however general time effects on body temperature of virus-inoculated ducklings were hard to discern . The wei10PFU/mL. The viremia titer with individual strains (10PFU/mL) were: KE-093-83 (4.1 ± 0.2), MAR864 (3.3 ± 0.2), JE-91 (4.2 ± 0.1), CH392 (3.0 ± 0.8), JKT27-087 (4.2 ± 0.3), and Sagiyama (3.3 ± 0.6). Similarly, the mean peak viremia titer  in the ducklings for G-I and G-III were 3.9 ± 0.2 and 3.5 ± 0.3, respectively. The mean peak viremia titers of ducklings were neither significantly different among the six strains , nor were they significantly different between genotypes . Virus was not detected after 3 dpi in any of the ducklings, nor in either of the two non-inoculated control ducks.The back-titrated dose of virus inoculated into ducklings ranged from 5.5 to 6.0 log strains  showed c10PFU/mL) was observed on 3, 4, and 5 dpi , JE-91 (2.5 ± 0.2) and JKT27-087 (3.9 ± 0.3), 4 of 5 ducklings inoculated with MAR864 (2.9 ± 0.2) and Sagiyama (3.7 ± 0.1), and 2 of 5 ducklings inoculated with CH392 (3.6 ± 0.2) shed viruses orally on 3 dpi. On 4 dpi, 1 out of 5 ducklings inoculated with KE-093-83 (2.0), JE-91 (3.2), and CH392 (3.5); 2 out 5 ducklings inoculated with Sagiyama (3.2 ± 0.7); and 4 out 5 ducklings inoculated with MAR864 (4.0 ± 0.3) and JKT27-087 (3.3 ± 0.3) shed viruses orally. On 5 dpi, only a single duckling from KE-093-83, MAR864 and JKT27-087 shed viruses orally of titer 2.5, 3.1, and 3.4 log10PFU/mL, respectively.Virus shedding in oral and cloacal secretions at and above the limit of detection , JE-91 (2.3), and JKT27-087 (3.3), and 2 out of 5 ducklings inoculated with KE-093-83 (2.3 ± 0.3) shed viruses cloacally.Regarding cloacal shedding of virus strains from ducklings, 1 out of 5 ducklings inoculated with CH392 (3.2), 2 out of 5 ducklings inoculated with KE-093-83 (3.4 ± 0.9) and MAR864 (2.2 ± 0.2), 3 out of 5 ducklings inoculated with JE-91(2.5 ± 0.3) and JKT27-087 (3.6 ± 0.1), and 4 out of 5 ducklings in Sagiyama (3.9 ± 0.2) shed viruses through cloaca on 3 dpi. On 4 dpi, cloacal shedding of virus was observed in ducklings inoculated with KE-093-83 only, and that 2 out of 5 ducklings shed the virus at a titer of 3.4 ± 0.7 log90 titer <10) before inoculation with the viruses as demonstrated by the absence of virus-specific antibodies. All ducklings that were inoculated with virus, except one in the CH392 group, seroconverted by 7 dpi, and the neutralizing antibody titer (PRNT90) increased by 21 dpi , infection percentage  and transmission percentage (2-tailed p = 0.0004) were significantly different between G-I and G-III on 7 dpf in Cx. quinquefasciatus. However, on 14 dpf, infection percentage , dissemination percentage , and transmission percentage (2-tailed p = 0.051) were not significantly different between G-I and G-III. Although, the effect of genotype x dpf on infection percentage  and dissemination percentage  was not significant, the effect was significant on transmission percentages . Our findings demonstrated JEV G-I is relatively better at infecting mosquitoes, disseminating through body parts of mosquitoes and being shed through saliva at an earlier time point.To test the hypothesis that G-I displaced G-III because it is more infectious for 10PFU/mL . Cx. qui strains . InteresCulex species mosquitoes [10PFU/mL on 2–3 dpi in Vero cells. Ducklings inoculated with freshly-grown viruses did not show signs of disease, but viremia was detected in serum obtained from all except 3 ducklings  on 1 to 3 dpi. Viremia ranged from 2–5.5 log10PFU/mL; previous studies indicated that only 10 PFU of JEV is enough to infect mosquitoes [Avian hosts are critical sources of JEV in endemic areas ,41,42, asquitoes . In the squitoes ,47. TherAll but one duckling seroconverted by 7 dpi and had higher antibody titer on 21 dpi, regardless of inoculated strain of JEV. Consistent with the demonstration that JEV G-III strain vaccine elicits higher antibody titer than JEV G-I strain vaccine in mouse model  in pigs Cx. quinquefasciatus mosquitoes are known to be competent vectors for JEV in several areas of Asia [Cx. quinquefasciatus in vivo [Cx. quinquefasciatus. Since, JEV infection in Vero cells inoculated with 7 dpi saliva content of mosquitoes which were blood-fed with JEV G-III viruses was not detected, our study suggests a difference in extrinsic incubation period in mosquitoes as a responsible factor in genotype displacement. A shorter extrinsic incubation period of WNV 02 genotype strains of West Nile virus was reported to be the cause of WNV NY99 genotype displacement in the US [ of Asia ,48, and  of Asia  and Cx.  in vivo . JEV G-In the US ,33. Genon the US ,33, DENVn the US , strain n the US , clade dn the US  demonstrIn addition to differences in EIP and host competence, other factors that may have contributed to genotype displacement include: (i) adaptation to the specific virus variants of mosquito species prevalent in different regions of Asia, (ii) the introduction of modernized farming practices including management of rice paddy fields and isolation of pig farms from urban environments, (iii) the implementation of concerted JEV immunization programs across many Asian countries, and (iv) the possible impact of climate change on migratory bird patterns and mosquito distribution. Although these represent possible mechanisms for displacement, evidence for those mechanisms are lacking.Cx. quinquefasciatus instead of known principle vector of JEV, Cx. tritaeniorhynchus, due to unavailability of the latter vector in the US. The Cx. quinquefasciatus used in this study have been maintained in a colony for several decades, and we understand vector competence for flaviviruses is impacted by a long-term colonization of mosquito vectors [Cx. quinquefasciatus [Cx. quinquefasciatus.Several caveats to the current study must be acknowledged. First, we utilized the Indian runner ducks in our study for several reasons: (i) they are a common domesticated breed of duck throughout Asia ,50,51,52 vectors . Howeverasciatus  nonethelCulex tritaeniorhynchus from areas where displacement has been recorded, and evaluate vectorial capacity, extrinsic incubation period and virus titers in mosquitoes with single and mixed genotype infection. However, such mixed infections of JEV G-III and JEV G-I may not occur at a high rate  in the field environment. In other words, competition may be demonstrated in the laboratory, but may be insignificant in the field since the frequency of mosquitoes being co-infected by both genotypes may not be sufficiently high to influence the predominant genotype. Host age is another feature that should be evaluated. The young ducklings used in the current study likely were not fully immunocompetent and differences among viral genotypes may have been masked at that young age.Future studies should include The possibility of potential impact of the concerted immunization campaigns against JEV which may have occurred when G-III was pre-eminent, that is, G-III would have been affected by the vaccine leaving a void for G-I to fill. In this context, the timeframe in phylogenetic terms  should be considered in future studies. It is possible that the presence of G-III as the predominant genotype for many years, coupled with vaccination using a G-III-based vaccine (SA-14-14-2) resulted in a high level of immunity to G-III in the community that was suboptimal in cross-protecting against G-I strains, facilitating establishment of G-I strains .The broader impact of our study is that exploration of host-virus interaction by the utilization of ecologically relevant hosts and vectors will elucidate the mechanistic basis behind genotype emergence and displacement. Viral and host ecology are key components of disease emergence and spread, and this finding can extend into an exploration of other zoonotic viruses that co-circulate, co-evolve and show displacement events. Our study substantially lends empirical support to the potential importance of variation in EIP as an explanation for genotype displacement in JEV, and will allow theoreticians to formulate a mathematical model to explore the impact of this variation at a population level. Moreover, the widespread and close proximity of domestic ducks and humans throughout Asia suggests that if ducks indeed serve as an important reservoir for JEV, they may have an impact similar to pigs in amplifying the virus. Clearly, a one health approach to control of this pathogen will be critical to formulating effective control strategies.


Escherichia coli (E. coli) and somatic coliphage are 7–9 times more likely to be associated with culturable virus-positive samples when the indicator is present versus when it is absent, while F-specific and somatic coliphages are 8–9 times more likely to be associated with culturable virus-positive wells. However, single indicators are only marginally associated with viruses detected by molecular methods, and all microbial indicators have low sensitivity and positive predictive values for virus occurrence, whether by culturable or molecular assays, i.e., indicators are often absent when viruses are present and the indicators have a high false-positive rate. Wells were divided into three susceptibility subsets based on presence of (1) total coliform bacteria or (2) multiple indicators, or (3) location of wells in karst, fractured bedrock, or gravel/cobble settings. Better associations of some indicators with viruses were observed for (1) and (3). Findings indicate the best indicators are E. coli or somatic coliphage, although both indicators may underestimate virus occurrence. Repeat sampling for indicators improves evaluation of the potential for viral contamination in a well.Groundwater quality is often evaluated using microbial indicators. This study examines data from 12 international groundwater studies (conducted 1992–2013) of 718 public drinking-water systems located in a range of hydrogeological settings. Focus was on testing the value of indicator organisms for identifying virus-contaminated wells. One or more indicators and viruses were present in 37 and 15% of 2,273 samples and 44 and 27% of 746 wells, respectively.  Groundwater is an important source of drinking water in both developed and developing countries. It constitutes about 95% of the world’s accessible freshwater  coliform bacteria, Bacteroides have also been evaluated as useful indicators  are present in wastewater and have been suggested to be a useful conservative indicator of fecal and viral pollution of groundwater , and survival characteristics of viruses differ fundamentally from those of bacteria -positive sample. The RTCR requires regular monitoring of all public systems for total coliforms using 100-ml samples at a frequency that depends on the population served. Any regular monitoring sample that contains any amount of total coliforms must be followed by additional tests for both total coliforms and E. coli. The compliance monitoring provision of the GWR requires that systems that provide 4-log treatment of viruses must conduct compliance monitoring to demonstrate continual treatment effectiveness. The GWR uses a risk-targeted approach to address the likelihood of viral contamination of wells—i.e., well susceptibility to contamination . It suggests that the agencies that implement the rule consider groundwater from aquifers in limestone, igneous and metamorphic rock, and gravel as potentially susceptible. However, among aquifers in gravel settings, gravel/cobble aquifers are more susceptible than those consisting of gravel/sand , these types of correlations are not always observed in groundwater  and USEPA conducted a study of viruses in groundwater in the US public systems during 1992–1994 Dahling , b. ThisThe US Geological Survey (USGS) and Public Drinking Water Program of the Missouri Department of Natural Resources conducted a two-phase study of 182 public water supplies in the Ozark plateaus aquifer system in Missouri during 1997–1998  during 2004  was conducted by the Marshfield Clinic Research Foundation in Wisconsin (USA) during 2006–2007  initiated several groundwater monitoring studies in metropolitan areas and provinces during 2007–2011  was conducted in the spring of 2013  between concentrations of viruses and indicators were evaluated at two levels, sample and well. By well, the sum of virus concentrations was compared to the sum of indicator concentrations. As many samples were negative for virus or indicator  which might result in spurious correlations, the analyses were repeated using a dataset restricted to culturable virus and PCR-virus positive samples.E. coli, enterococci, F-specific phage, somatic phage, spores (anaerobic spores + aerobic spores)—was treated as a dichotomous variable (detect or non-detect) and compared to two measures of virus contamination: culturable virus and PCR-virus. Both outcome measures were also treated as dichotomous variables . The strength of the indicator-virus association additionally was quantified by calculating the risk ratio . The null value for the risk ratio is 1.0 with values greater than 1.0 representing the relative elevation in the virus detection rate if the indicator is detected versus when the indicator is not detected. The preceding analyses were conducted separately for data at the level of the sample and well and all results were derived from logistic regression models.The utility of water quality indicators as indicators of virus-contaminated groundwater was evaluated by testing the association between indicators and virus and by calculating the four conventional performance measures of a diagnostic test: sensitivity (i.e. true positive rate), specificity , positive predictive value, and negative predictive value  and study only . These models also incorporated robust variance estimation , since this could affect the corresponding probability of detection for the well; however, this resulted in model convergence problems. Within each study NTWS was generally homogeneous, either a small number of similar values or dominated by a single value. It is possible that the model adjustment for study lessened any effect of NTWS, but this may be a limitation of the well-level analyses.Table P < 0.001, n > 1,300) for combinations of total coliforms, E. coli, and enterococci, and between enterococci and somatic phage (data not shown). Between the indicators and culturable virus concentrations on a per-sample basis the trends were positive but weak . Correlations among indicators and PCR-virus  and somatic coliphage  on a per-sample basis and somatic coliphage  on a per-well basis; however, restricting the data set did not improve the correlations among the indicators and virus detected by molecular assays (data not shown).The analysis was repeated by restricting the data to virus-positive samples (or wells) to minimize the effect of non-detects on the correlations. With this restricted data set, the highest correlations to culturable virus were E. coli, and somatic phage), were significantly associated with PCR-viruses. The two most commonly used indicators in the US—total coliforms and E. coli—were associated at the well level with both culturable viruses and PCR-viruses, albeit the E. coli and culturable virus association was marginally not significant  using logistic regression models and the data from the 12 studies. Culturable viruses were associated at statistically significant levels with the indicators except for total coliforms measured at the sample level and spores measured at the well level Table . In contE. coli sample is associated with a seven-times greater chance of detecting culturable viruses in a corresponding sample compared to a negative E. coli result. For PCR-viruses, the chance of detection in a sample is similar whether a corresponding E. coli sample is positive or negative . However, at the level of well, an E. coli positive well is associated with a 60% greater chance the well, at some time, will be positive for PCR-viruses .The risk ratios reported in Table Table Indicator test performance measures were examined next. Sensitivities of the indicators for signifying whether a virus was detected in a sample or well were low (2–30%), with estimates being relatively higher when virus positivity was determined by culture . Positive predictive values at the well level were higher (but still mostly <50%) than at the sample level. Approximately 30 to 40% of the wells that were positive for an indicator were contaminated with virus at some point during the sampling period.E. coli had 95 and 97% specificity at the sample level for culturable virus and PCR-virus, respectively. In other words, a negative E. coli result correctly identified 95–97% of the samples that were negative for viruses. Specificity values at the well level were consistently lower than at the sample level for all six indicators, but the magnitude of the differences were usually not large.In contrast, specificities of the indicators were high, with values often near 90% or greater for culturable virus and PCR-virus. For example, Negative predictive values for the indicators were generally greater than 90%, with the exception of PCR-viruses at the well level, where estimates ranged from 50 to 76%. An indicator with a high negative predictive value means that when a sample or well is indicator-negative, it is likely to be also virus-negative .The sample and well data were reanalyzed according to the TCR, Hydrogeology, and GWR susceptibility subsets described in Table Table This study addresses the relationship between microbial indicators and human enteric virus in groundwater. The focus of the study is on public systems in the US, Canada, Europe, and Asia for which raw data from 746 wells and 2273 samples were available. One or more indicator was found in 44% of the wells, while culturable virus was detected in 8% and PCR virus in 30% of the wells.Many studies have examined correlations between concentrations of indicators and viruses and often times the correlations are weak or non-existent  while adjusting the association for underlying differences among the 12 studies and among wells within a study. Risk ratios and tests for association evaluate the overall correspondence between indicator and virus occurrence. A more complete interpretation of an indicator’s value with respect to viruses is provided by calculating standard test performance measures  that convey the likelihood of false positives and false negatives . Additionally, a positive indicator result does not necessarily mean the well is virus-contaminated; it could likely be a false positive . If the well does not have a virus contamination problem, there is a reasonable chance the indicator results will be negative, confirming there is no problem (high specificity). And a well that is indicator negative is unlikely to be virus contaminated, especially for culturable viruses . In summary, the downside of the indicators is that many virus positive wells can be missed and there could be many false positives. On the other hand, wells that are indicator-negative are unlikely to have virus contamination problems. Moreover, sensitivities and positive predictive values at the well level were higher for all six indicators compared to the sample level, for the most part without sacrificing a large drop in specificity or negative predictive value. This suggests that multiple samples from a well improves indicator performance in assessing a well’s susceptibility to virus contamination. Indicator performance might also be improved by considering not just the detection of the indicator, but also its concentration. Payment and Locas  showed tThe indicator-virus associations were evaluated for three well susceptibility categories to examine if indicators were more informative based on prior knowledge of well-specific data Table . The TCRSensitivities, positive predictive values, and risk ratios tended to be higher for total coliforms, enterococci, F-specific phage, and somatic phage when they were measured in samples or wells in the TCR or hydrogeology susceptibility categories. In other words, for wells with two or more TCR violations or wells located in susceptible hydrogeological settings, like karst, these indicators performed better at identifying samples and wells that were true virus-positives and with a lower false positive rate. However, for wells that would have met the criteria under the GWR for additional monitoring, none of the single indicators performed any better than when they were measured in all wells regardless of susceptibility status.E. coli, enterococci, aerobic spores, F-specific and somatic coliphages. Standard methods should be used as much as possible.In interpreting the data presented in the preceding, it needs to be stressed that there is a degree of uncertainty in the placement of wells into the susceptibility categories, particularly the hydrogeology category. Interpretation is weakened by the lack of uniformity in the types of stressors measured by individual studies as well as by the different methodologies used. Future studies that examine indicator and virus relationships should measure viruses by both culture and molecular assays, and include at least total coliform, The finding of a virus by either culturable or molecular assays is not always easily translatable to the public health risk presented by a particular well. Virus concentration in groundwater can vary rapidly over time . None of the indicators were associated with PCR-measured viruses at the sample level, and only total coliforms,


Plants are persistently challenged by various phytopathogens. To protect themselves, plants have evolved multilayered surveillance against all pathogens. For intracellular parasitic viruses, plants have developed innate immunity, RNA silencing, translation repression, ubiquitination-mediated and autophagy-mediated protein degradation, and other dominant resistance gene-mediated defenses. Plant viruses have also acquired diverse strategies to suppress and even exploit host defense machinery to ensure their survival. A better understanding of the defense and counter-defense between plants and viruses will obviously benefit from the development of efficient and broad-spectrum virus resistance for sustainable agriculture. In this review, we summarize the cutting edge of knowledge concerning the defense and counter-defense between plants and viruses, and highlight the unexploited areas that are especially worth investigating in the near future. Plant viruses are obligate intracellular parasites that rely almost exclusively on the host cell to accomplish their infection cycle, e.g., genomic information expression, genome replication, and intercellular movement. During the infection, a comprehensive and competitive protein–protein and protein–nucleic acid interaction network is formed between the plant and virus. In general, the interaction network includes mechanisms by which the plant forms antiviral responses and the virus hijacks host factors for proliferation and to cope with plant antiviral defenses. The elaborate balance between the mechanisms used by the plant and the virus determines the outcome of the interaction, whether a virus is pathogenic or not. Understanding the plant–virus interaction network is one of the key goals of virology research. Impressive progress has been made in the knowledge of plant–virus interplay in the past decade, which not only quickly filled in the gap between plant innate immunity against virus and other pathogens, but also revealed unique plant antiviral machinery, such as NUCLEAR SHUTTLE PROTEIN (NSP) INTERACTING KINASE 1 (NIK1)-mediated translation repression and atypical dominant resistance by inhibiting viral protein function. In this review, we present the cutting edge of knowledge concerning the defense–counter-defense between plants and viruses, and outline the areas that may be particularly fruitful for study in the coming years. Due to space limitations, how plant viruses hijack the host’s metabolism for multiplication and dissemination is not included in the review, and readers are referred to the recent reviews . RecessiPlants are persistently challenged by varied pathogens, including viruses, bacteria, fungi, nematodes, and insects. Unlike the animal counterparts that possess specialized defense cells, plants rely on the capacity of every cell to perceive and defend against the “challengers”. During the millions of years of co-evolution with pathogens, plants have evolved multilayered surveillance mechanisms against pathogens that invade them ,7. InnatPR) genes, the synthesis and deposition of callose at the plasmodesmata (PD), and strengthening of the cell wall [According to current plant immunity descriptions, there are two layers of plant immune responses against microbial pathogens. First, plant perception of the microorganism at the cell surface through the recognition of certain conserved pathogen-associated molecular patterns (PAMPs) by the extracellular surface pattern recognition receptor (PRR) initiates the so-called PAMP-triggered immunity (PTI) ,10. Planell wall ,14. Occaell wall . At presell wall .Capsicum annuum L.), results in the enhanced susceptibility of pepper to several unrelated pathogens, including two plant viruses, tobacco mosaic virus , and pepper mild mottle virus , one bacterium (Xanthomonas campestris pv. vesicatoria), and one oomycete pathogen (Phytophthora capsici) [Despite the mass of knowledge on plant PTI against other phytopathogens, little is known about plant PTI against plant viruses, as they are historically viewed as non-PAMP coding pathogens . Neverthcapsici) . Moreovecapsici) , restorecapsici) . Second,capsici) ,21, alsocapsici) . Third, capsici) ,24. CP icapsici) . NeverthR genes that mediate resistance against various plant viruses have been cloned, as they have apparent practical application value [R genes, such as the potato Rx1 gene, confer an extreme immunity that is not associated with HR [N gene [Adapted pathogens can deliver specific proteins (effectors) into plant cells to compromise PTI defenses. To further counteract the effectors, plants have evolved an additional monitoring system that relies on particular intracellular receptors known as R proteins to directly or indirectly recognize the microbial effectors that are used to subvert PTI and trigger the so-called effector-triggered immunity (ETI) . Over thon value ,28. Funcon value . In addion value . The NB-on value . Similaron value . However with HR . Rx1 rec with HR ,34,35. T with HR . However [N gene ,38,39.N-mediated and Rx-mediated resistance against TMV and PVX, respectively, and immune responses against bacterial infections [The components that are required for R-mediated resistance against virus infection largely overlap with those for other phytopathogens, suggesting the convergence of plant ETI . For insfections ,41,42,43fections ,46,47,48PR) genes [NPR1 attenuates PR gene expression [Both PTI and ETI can result in the establishment of resistance in the distal, non-infected tissues, which is a phenomenon called systemic acquired resistance (SAR) . IntriguR) genes . HoweverR) genes . Neverthpression . Thus, Npression .RNA silencing, which is also called RNA interference (RNAi), is an evolutionary conserved and sequence-specific mechanism for regulating endogenous gene expression and fighting against foreign nucleic acids such as transposable elements and viruses . The virPR gene expression and HR development, and is independent of small molecules, e.g., SA, reactive oxygen species (ROS), or calcium ions, as secondary messengers for signal amplification.Similar to innate immunity, RNA silencing also (1) perceives extraneous dsRNA molecules in a nucleotide sequence-independent manner; (2) conducts a signal amplification process; and (3) establishes resistance against homologous virus infection on non-infected tissues via the cell-to-cell and systemic movement of vsiRNA duplexes ,71. HoweThe cellular protein biosynthesis is tightly regulated in response to nutrient starvation or stresses. It is believed that GENERAL CONTROL NON-DEREPRESSIBLE-2 (GCN2), which is a serine/threonine-protein kinase, is the key regulator of protein biosynthesis in plants. GCN2 functions by targeting and phosphorylating EUKARYOTIC TRANSLATION INITIATION FACTOR-2A (eIF2α), which binds Met-tRNA and transfers it to the 40S ribosomal subunit to initiate the synthesis of a protein upon bonding to guanosine triphosphate (GTP) ,73. The Phytolacca americana). PAP has been demonstrated to be able to reduce the propagation of many plant viruses, e.g., cucumber mosaic virus , PVX, tobacco etch virus , alfalfa mosaic virus , potato virus Y , brome mosaic virus , African cassava mosaic virus , cauliflower mosaic virus  [Ribosome-inactivating proteins (RIPs) form a family of proteins that is ubiquitously distributed in plants, mushrooms, and bacteria and can inhibit protein synthesis by depurinating the sarcin/ricin loop (SRL) of rRNA ,81. Resemovirus) ,84,85,86movirus) . Moreovemovirus) , suggestmovirus) . How to PR genes; instead, NIK1 phosphorylates the cytoplasmic RIBOSOMAL PROTEIN 10 (RPL10) and results in the translocation of RPL10 from the cytoplasm to the nucleus [Recently, another antiviral mechanism that functions by suppression of the global cellular protein synthesis was discovered . NUCLEAR nucleus . In the  nucleus .Besides global translation repression, plants also have developed specific translation repression machinery. One such example is small RNA-related translation repression. As addressed in the above content, the binding of miRNA-associated and short interfering RNA (siRNA)-associated RISCs to cellular or viral RNA, in addition to endonucleolytic cleavage, also causes direct translation inhibition ,62,95,96The exploration of viral resistance in the last decade also resulted in the discovery of some dominant resistance genes that are independent of the typical innate immunity signaling cassette for function. The products of these dominant resistance genes are structurally different from each other and from the typical R proteins, and, thus cannot be integrated into the conventional plant innate immunity. Based on the current understanding, most of these dominant resistance genes likely function by directly interacting with viral proteins to inhibit their activity. Hereafter, we referred to the products of these dominant resistance genes as ATYPICAL DOMINANT VIRAL RESISTANCE PROTEINs (ADVRPs).RTM4 and RTM5 genes, which were only genetically characterized [Cyamopsis tetragonoloba (L.) that is able to confer tobacco with resistance against the Sunn-hemp mosaic virus  and a lectin from Musa paradisiaca (BanLec-1) that binds to the CP of TMV and prevents viral infection [The major ADVRPs discovered so far are members of the lectin protein family. Lectins, which are also known as agglutinins or jacalins, are a group of the carbohydrate-binding domain (CBD)-containing proteins that bind reversibly to specific monosaccharides or oligosaccharides . Besidescterized ,108,109.cterized . JACALINcterized ,112,113.nfection ,115. Ty-1 and Ty-3 encode a γ-class RdRp that confers resistance to tomato yellow leaf curl virus , and the hypermethylation of the genomic DNA has been hypothesized as the possible source of the resistance [Other ADVRPs, such as the tomato Tm-1, which confers resistance to tomato mosaic virus   and VIOLsistance ,121.As research progresses, the number of ADVRPs will continue expanding. However, the origin of these ADVRPs is still a mystery at the present. A likely possibility is that these proteins have primary functions that are other than antiviral, and incidentally acquired the ability to inhibit virus proliferation . For insPR genes’ expression in plant innate immunity involves the proteasome-mediated turnover of the transcription co-activator NPR1 [Ubiquitination-mediated protein degradation is the major protein turnover machinery in the plant. Ubiquitination is a multistep enzymatic reaction that is able to covalently attach ubiquitin (Ub) to the target proteins . First, tor NPR1 . Furthertor NPR1 . Unfoldetor NPR1 .Nicotiana tabacum RING FINGER PROTEIN 1 (NtRFP1) interacts with the βC1 protein encoded by the tomato yellow leaf curl China virus (TYLCCNV)-associated betasatellite, which is a viral pathogenesis factor, and VSR [As the specificity of ubiquitination-mediated protein degradation is determined largely by E3, both plants and animals encode only a few E1s and E2s, and a large number of E3s. For instance, the model plant Arabidopsis encodes two E1s, 37 E2s, and more than 1400 E3s . The mas and VSR ,132,133, and VSR . Besides and VSR ,136, and and VSR ,138. HowAutophagy is conserved machinery for transporting unwanted or misfolded protein aggerates or damaged organelles to the vacuole for degradation and recycling. In plants, it is pivotal for maintaining cellular homeostasis under normal conditions and resisting abiotic and biotic stresses . SimilarR genes are regulated by microRNAs, e.g., miR472, and further targeted by the trans-acting siRNAs (ta-siRNAs) or siRNAs produced from their transcripts by plant RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and the expression of the components of the RNA silencing pathway are also regulated by innate immunity responses [Plant antiviral mechanisms do not function separately; in contrast, there is vigorous cross-talking between different defense programs. In general, there are three types of cross-talking. Firstly, the component of one antiviral pathway is always regulated by other antiviral pathways. For instance, the transcripts of esponses ,149,150.esponses ,151,152.esponses . SA is aesponses . A calmoesponses ,156,157.Plant viruses have also gained multiple strategies to suppress and even exploit host defenses to ensure their successful infection . DespiteRice yellow mottle virus  results in the loss of resistance of the Oryza glaberrima Tog7291 line carrying the RYMV2 resistance gene [Rz1-mediated resistance against Beet necrotic yellow vein virus  infection can be caused by a single mutation in its p25 coding sequence [Due to the lack of proofreading viral RdRp, both plant and animal RNA viruses have a much higher mutation rate than that of their host cell, which is replicated via the DNA polymerase ,159. Plance gene . The bresequence . A singlsequence ,164. AchDespite the wealth of knowledge on how other phytopathogens and animal viruses suppress host immune responses ,165, howTombusvirus within the family Tombusviridae, have very a similar genome structure, particle morphology, and replication strategy. However, these two viruses utilize the outer membrane of mitochondria and peroxisomes for replication, respectively [Potyviridae), TMV (family Virgaviridae), and BMV (family Bromoviridae) hold replication vesicles or organelle-like structures derived from the ER [Antiviral RNA silencing mainly occurs in the cytoplasm, where the replication of most plant RNA viruses takes place. To encounter host RNA silencing, the replication of many positive-sense single-strand RNA (+ssRNA) viruses are concealed in membranous inclusion bodies, vesicles, multivesicular bodies, or spherules that are remodeled from the plant cell endogenous membranes by viral proteins ,174,175.ectively ,177. Meam the ER ,179,180.m the ER . Interesm the ER ,185,186,Membranous virus replication factories are not sufficient to support robust viral replication, as abnormally abundant viral RNA in the plant cell can also trigger RNA silencing through the de novo synthesis of dsRNA by the host RdRp, which in turn inhibits viral protein synthesis and viral particle assembly. For instance, a CMV mutant that lacks the protein suppressing host RNA silencing (2b), can only replicate and cause systemic infection with extremely low efficiency ,188. RemAGO1, which is a target of miR168 [Plant viruses can also exploit the host RNA silencing system to promote their replication. Plant viruses exploit host RNA silencing mainly in two ways: one is by regulating endogenous miRNA expression, and another is by suppressing endogenous gene expression through viral-derived small-interfering RNAs (vsiRNAs). One such example is the infection of cymbidum ringspot virus , whose VSR (p19) can specifically upregulate the expression of miR168 to downregulate the transcripts of f miR168 . A similf miR168 . Recentlf miR168 . Indeed,f miR168 ,200,201.f miR168 ,202.A substantial amount of evidence suggests that animal viruses are able to subtly manipulate host protein synthesis machinery and adopt numerous unconventional translation mechanisms for a productive proliferation ,204. PlaNSP is the geminivirus-encoded membrane protein that counteracts NIK1-mediated translation repression . NSP dirPlant viruses also have developed several strategies to subvert host ubiquitination-mediated and autophagy-mediated antiviral machinery, e.g., by encoding deubiquitinases that target different endogenous and viral proteins . For insPlant viruses are able to exploit ubiquitination and autophagy to promote their proliferation by encoding ubiquitin ligases or molecular adaptor proteins that target specific host factors for degradation. A large number of such examples have been reported, including the P0 protein of poleroviruses and enamoviruses, an F-box domain-containing protein, and PVX TGBp1 target AGO proteins for degradation via ubiquitin-mediated proteasome and autophagy pathways ,213,214;R gene-directed breeding slowed down the ravages of many destructive plant viruses, such as tospoviruses, potyviruses, and begomoviruses [Compared to the plant defense against other extracellular micropathogens, e.g., fungi and bacteria, in some aspects, plant antiviral defense could be more complex. This is apparent due to the intracellular parasitism of plant viruses, in which all genetic materials are directly explored by and connected with plant intracellular factors. Although this direct interaction allows the plant to evolve new defense mechanisms targeting viral factors, plant viruses can also take advantage of the interaction to explore the weak points of plant antiviral barriers and exploit their error-prone polymerases and multiple functional proteins to quickly escape host defenses and win the arms race. The ultimate goal of plant–virus interaction research is to establish sustainable virus resistance strategies to ensure food safety for the expanding human population. Indeed, considerable successes have been made in the management of viral diseases for varied crops in the last decade. For instance, oviruses ,228. Grooviruses ,230. Smaoviruses . Althougoviruses ,236,237,oviruses ,244,245.Sw-5 gene  with to epitope . Thus, t


Aedes (Stegomyia) species. In 2013–2014, Ae. aegypti transmitted CHIKV to humans in the Caribbean and in 2005–2006, Ae. albopictus transmitted CHIKV on La Réunion Island (Indian Ocean basin). CHIKV LR2006 OPY1 from the La Réunion epidemic was associated with a mutation (E1:A226V) in the viral E1 glycoprotein that enhanced CHIKV transmission by Ae. albopictus. CHIKV R99659 from the Caribbean outbreak did not have the E1:A226V mutation. Here, we analyzed the salivary glands and saliva of Ae. albopictus strains from New Jersey, Florida, Louisiana and La Réunion after infection with each virus to determine their transmission potential. We infected the Ae. albopictus strains with blood meals containing 3–7 × 107 PFU/mL of each virus and analyzed the mosquitoes nine days later to maximize infection of their salivary glands. All four Ae. albopictus strains were highly susceptible to LR2006 OPY1 and R99659 viruses and their CHIKV disseminated infection rates (DIR) were statistically similar (p = 0.3916). The transmission efficiency rate (TER) was significantly lower for R99659 virus compared to LR2006 OPY1 virus in all Ae. albopictus strains and Ae. aegypti (Poza Rica) (p = 0.012) suggesting a salivary gland exit barrier to R99659 virus not seen with LR2006 OPY1 infections. If introduced, LR2006 OPY1 virus poses an increased risk of transmission by both Aedes species in the western hemisphere.Chikungunya virus (CHIKV) is a medically important mosquito-borne virus transmitted to humans by infected  Chikungunya virus  is an arthropod-borne virus (arbovirus) maintained during epidemics in transmission cycles between viremic humans and infected Aedes (Stegomyia) mosquitoes. The genome of CHIKV is a positive single stranded RNA  containing two open reading frames (ORFs) between the 5′ and 3′ untranslated regions [9 viral genome copies per milliliter of blood or virus titers of 107–9 plaque-forming unit equivalents /mL [Ae. aegypti, a peridomestic, day-biting mosquito found in tropical and subtropical regions of the world [Ae. albopictus [Ae. albopictus has invaded new regions globally. They now thrive in temperate areas of the western hemisphere and could pose additional health risks to a CHIKV susceptible human population [ regions . The onsents /mL ,3. The phe world . Howeverbopictus . SignifiAe. albopictus [Ae. albopictus virus can be further enhanced by amino acid changes in their E2 glycoprotein [Ae. albopictus populations. Autochthonous transmission of ECSA-IOL CHIKV by Ae. albopictus could then lead to new outbreaks in CHIKV-naïve human populations [CHIKV are classified in three genotypic groups: East-Central-South Africa (ECSA), West Africa, and Asian. After the 2006 La Réunion outbreak, a new Indian Ocean lineage (IOL) was identified as a monophyletic descendant of the ECSA group ,8. The Rbopictus ,10,11,12oprotein . In 2013oprotein ,15,16. Tulations .Ae. albopictus from New Jersey, Florida and Louisiana. We compared infections of these mosquitoes with an Ae. albopictus strain from La Réunion Island and an Ae. aegypti strain from Mexico (Poza Rica). The extrinsic incubation period (EIP), the time from initial acquisition of CHIKV by the mosquito until virus transmission ranges from 2 to 9 days [Ae. albopictus had high DIR values. The TER values for CHIKVs were lower than expected for R99659 virus given the mosquitoes’ high DIRs and suggested the presence of a salivary gland escape barrier (SGEB) for that virus. All mosquitoes were competent to transmit LR2006 OPY1. Of the three U.S. Ae. albopictus strains, the Louisiana and Florida strains could serve as excellent vectors for CHIKV R99569 (Asian) transmission. The New Jersey Ae. albopictus strain had the lowest vector competence (DIR × TER) of the three Ae. albopictus strains for R99569 virus but had statistically similar vector competence as the other strains for LR2006 OPY1 (ECSA-IOL) [Our goal was to analyze salivary gland and the titer of virus in saliva of the two CHIKV isolates, LR2006 OPY1 (ECSA-IOL genotype) and R99659 (Asian genotype), in strains of o 9 days . We analCSA-IOL) . A. albopictus) cells were cultured in modified Eagle’s medium (MEM) supplemented with 8% fetal bovine serum, l-glutamine, non-essential amino acids and penicillin/streptomycin and maintained at 5% CO2 at 37 °C and 28 °C, respectively. The two low passage CHIKV isolates  and LR2006 OPY1  were provided by the Centers for Diseases Control and Prevention . The presence of the E1-A226V mutation in LR2006 OPY1 virus was confirmed by RT-PCR using specific primer sets designed to amplify the nucleotide sequence surrounding the mutation as previously described [Vero cells, LLC-MK2 monkey kidney cells and C6/36 , and La Réunion Island (Indian Ocean) were provided by the Centers for Diseases Control and Prevention . The New Jersey Ae. albopictus strain was obtained from Cornell University and the Ae. aegypti line was collected in Poza Rica, Mexico [, Mexico ,21. The , Mexico . Blood e2O): nutrient solution , 7.5% sodium bicarbonate (4mL/100mL), 2% DEAE- dextrose in Hanks balanced solution (1mL/100mL), 0.5 mL/100mL MEM essential amino acids (15× solution), and 0.5 mL/100mL MEM vitamins (100× solution)). After 7 days incubation at 37 °C cells were stained with 3 mg/mL MTT -2,5-diphenyltetrazolium bromide) solution and incubated for 4 h [Plaque assays were performed using confluent monolayers of Vero cells in 24-well plates to determine virus titers in saliva and salivary glands. The samples were sterilized by passing them through Acrodisc HT Tuffryn 0.2-μm syringe filters . Vero cells were infected for 1 h with 10-fold serial dilutions of saliva or salivary gland homogenate samples. Infected cells were overlaid with a 1% agar-nutrient mixture  = 3.1416 × 0.01 × h = mm3 or µL). Following expectoration, salivary glands were dissected from the same mosquitoes that provided saliva and placed in 500 μL of DMEM medium . The salivary gland samples were frozen immediately (dry ice) prior to determining CHIKV titers. CHIKV titers in saliva and salivary glands were reported as pfu/mL from plaque assays as previously described. At day 9 post-infections, saliva from each female was collected using a previously described saliva collection method . Brieflyp < 0.05) in DIR and TER  and the correlation among virus titers in saliva and salivary glands (Pearson correlation). Analysis of variance (one-way and two-way ANOVA) was used to determine the statistical significance and CORREL function  for determining the correlation coefficient. The proportion of infected salivary glands and saliva were calculated by averaging the number of infected samples divided by the total tested. All data were analyzed with GraphPad prism software  for testing significant differences  or LR2006 OPY1 . We detected no significant difference in DIR among the Ae. albopictus strains  infected with either R99659 and LR2006 OPY1 (p = 0.3916). DIR ranged from 93% to 96.6% for R99659 and 93.3% to 100% for LR2006 OPY1. However, significant differences were detected in the virus titers of salivary glands among the Ae. albopictus strains infected with either R99659 (p < 0.0001) or LR2006 OPY1  or Louisiana strain (p = 0.0181). LR2006 OPY1 virus titers in the New Jersey strain were significantly lower than in the Florida and La Réunion strains of Ae. albopictus , Florida (p = 0.0089) and New Jersey (p = 0.0472) strains. We observed no significant differences in DIR between the two CHIKV isolates in the Louisiana strain (p = 0.1920).The DIR of Ae. albopictus strains infected with R99659 virus (p = 0.3916) or the Ae. albopictus strains infected with LR2006 OPY1 virus. The prevalence (TER) of virus in saliva ranged from 33.3% to 56.6% for R99659 virus and 80% to 86.6% for CHIKV LR2006 OPY1 virus. The TER of R99659-infected Ae. albopictus strains was significantly lower than mosquito strains infected with LR2006 OPY1 virus  possibly indicating LR2006 OPY1 virus has a fitness advantage in these mosquitoes.We did not detect significant differences in virus prevalence in saliva (TER) among the A. aegypti (Poza Rica) for R99659  or LR2006 OPY1  were also evaluated 9 days post-infection. DIRs were high for all CHIKV isolates tested in Ae. aegypti, with no significant difference (p = 0.3679) between R99659 and LR2006 OPY1 viruses (Ae. aegypti (Poza Rica) infected with LR2006 OPY1 and R99659 viruses were statistically similar (p > 0.05). These data indicated that Ae. aegypti (Poza Rica) strains had statistically similar vector competence (DIR × TER) for these viruses. DIR, TER and vector competence for the two CHIKVs and the four Ae. albopictus strains and one Ae. aegypti strain are summarized in The DIRs of  viruses . The DIR viruses . The TERAe. aegypti . Although the New Jersey A. albopictus strain had a positive correlation between the two variables, it was weak and likely insignificant  and saliva virus titer (pfu/mL) for R99659 and LR2006 OPY1 viruses in genotype B and the006 OPY1 C. HoweveAe. aegypti (Poza Rica) strain since many mosquitoes were without detectable virus in their saliva even though their salivary glands were highly infected and all mosquitoes were able to salivate regardless of their infection status. This was also true for the four Ae. albopictus strains tested but the correlation was weaker than the SGEBs associated with Ae. aegypti. Interestingly, SGEB was dependent upon the viral genotype, since the four Ae. albopictus strains and the Ae. aegypti strain infected with R99659 (Asian genotype) showed a higher SGEB than mosquitoes infected with LR2006 OPY1 virus. The New Jersey Ae. albopictus strain infected with R99659 virus presented the highest SGEB (64.3 %) compared with the Florida, La Réunion or Ae. aegypti (Poza Rica) strains  was determined by dividing the number of saliva samples without virus by the number of virus positive salivary glands. The highest SGEB occurred with the n = 30/group) for each virus. The four Ae. albopictus strains had significantly higher vector competence for LR2006 OPY1 virus versus R99659 virus  also had a significantly higher vector competence for LR2006 OPY1 virus than R99659 virus (p = 0.005). Comparisons of the vector competence of Ae. aegypti versus Ae. albopictus for LR2006 OPY1 were not different (p = 0.1881). Comparisons of Ae. aegypti (Poza Rica) versus the Ae. albopictus strains  for R99659 were also not statistically different (p > 0.05). The only significant differences when comparing vector competence of mosquito groups for R99659 virus was between the New Jersey strain and the Florida or Louisiana Ae. albopictus groups (p = 0.0191 and p = 0.0083).We multiplied DIR by TER to determine vector competence of each group of mosquitoes  prefilled with 1 µL of immersion oil type B. Twenty-six mosquitoes were allowed to salivate into the oil at room temperature for 1 h and then measured the height of saliva in the capillary tube with a digital fractional caliper (presicion: ±0.02 mm). The volume of saliva was then calculated using the cylinder volume formula (V = π(r2 × h) = 3.1416× 0.01 × h = mm3 or µL; Ae. aegypti mosquitoes salivated an average of 6.82 ± 2.88 nL  from hundreds of Ae. aegypti saliva samples [In our assay, saliva is expectorated just after the mosquito proboscis immerses into collection fluid. Mosquitoes such as  process . The rat 2.88 nL B. We the 2.88 nL C. Figure samples .Ae. albopictus and Ae. aegypti populations were performed here to indicate vector competence. We reasoned that the high DIR values ranging from 93% to 100% of infected salivary glands for each Aedes species lessened the importance of analyzing midgut infection rates 9 days after per os infection.Vector competence, the ability of an arbovirus vector to acquire a virus and successfully transmit the virus to another susceptible host, is determined by genetic traits of vector species and vector populations within species. Vector competence, the efficiency of viral replication and external factors including temperature, the availability of vertebrate hosts, vector population density and predation, determine how successful a vector population will be in transmitting virus in nature . DIR andAe. albopictus strains clearly had uniformly high DIR or CHIKV prevalence in salivary glands after infection with R99659 or LR2006 OPY1 virus. Although the DIRs were similar among the Ae. albopictus strains tested, the salivary glands infected with the two CHIKVs varied in virus titers. The mean virus titer of both CHIKVs were highest for the La Réunion strain and lowest for the New Jersey strain indicating that geographic origin of the vector is an important factor. Nevertheless, differences in vector competence of the New Jersey strain for LR2006 OPY1 were not significant when compared to the other Ae. albopictus strains and Ae. aegypti (Poza Rica)  see . CHIKV sof CHIKV ,31,32,33Ae. albopictus strains had statistically similar DIRs for both CHIKVs, the TER for CHIKV R99659 virus was significantly lower in all mosquito strains  than the LR2006 OPY1 virus (p = 0.012) suggesting the LR2006 OPY1 virus is more efficiently transmitted than R99659 virus. The New Jersey strain had lower TER values than the other Ae. albopictus strains tested suggesting that population was less able to transmit CHIKV. Our results support earlier reports that noted similar DIR and TER outcomes for CHIKVs in Ae. aegypti and Ae. albopictus populations originating in the Americas [Although the Americas . Ae. aegypti model that the volume of saliva does not influence virus titers in saliva  of E1 [Ae. albopictus for LR2006 OPY1 [Ae. albopictus to ensure the virus’ survival and transmission [We found no correlation between the virus titer in the salivary gland and virus titers in the saliva  and demon saliva . AlthougV) of E1 . CHIKV w006 OPY1 . Interes006 OPY1 . Given tsmission .Aedes vectors may lead to barriers to CHIKV transmission that vary with mosquito and viral genetics. Our data demonstrated that 93–100% of mosquitoes had salivary gland infections but only 33–86% of saliva samples were positive for the two CHIKVs. This difference was more pronounced in Ae. aegypti than Ae. albopictus. We also measured saliva volume from Ae. aegypti and saw little correlation between the volume of expectorate and virus titer. SGEBs have been reported for La Crosse (LACV) and Sindbis virus transmission by Aedes and Culex species, respectively [Aedes hendersoni was shown to be an incompetent vector of LACV due to a SGEB in which the salivary glands were infected but the mosquito failed to transmit the virus orally [Ae. aegypti [Ae. aegypti and Ae. albopictus vary according to the origins of mosquito populations tested and the CHIKV strain.The DIR and TER data suggests that the salivary glands of ectively ,37,38,39s orally . Moleculs orally ,42,43. C aegypti . We demoAe. albopictus and Ae. aegypti exists to transmit CHIKV to a susceptible human population. The emergence of mosquito-borne CHIKV in the Americas starting in 2013 led to a geographically widespread outbreak with human illness and the establishment of CHIKV transmission cycles in urban settings. As evidence of this, 11 cases from autochthonous transmission of CHIK were detected in south Florida in 2014 [CHIKV is now widespread worldwide and likely will continue to pose a public health threat globally wherever the invasive mosquitoes  in 2014  emphasiz(1)Ae. albopictus strains and Ae. aegypti with blood meals containing 5–7 × 107 PFU/mL of R99659 (Asian genotype) and LR2006 OPY1 (ECSA-IOL genotype) and analyzed the mosquitoes nine days later to detect maximum infection of their salivary glands and saliva.We infected the (2)Ae. albopictus strains were highly susceptible to LR2006 OPY1 and R99659 viruses and their CHIKV disseminated infection rates (DIR) were statistically similar (p = 0.3916).All four (3)Ae. aegypti and Ae. albopictus vary according to their geographic origins of the mosquitoes and the CHIKV strain contributing to a salivary gland barrier for releasing R99659 virus into saliva.We demonstrated here that the TER in (4)Ae. aegypti expectorated on average a volume of 6.8 nL/mosquito.Virus titers in salivary glands did not correlate with saliva volume, but (5)Aedes species in the western hemisphere.If introduced, LR2006 OPY1 virus could increase the risk of CHIKV transmission by both


In this population, we identified 149 putative instances of superinfection . Unexpectedly, we discovered a group of 240 individuals with consecutively sampled viral strains that were >0.015 substitutions/site divergent, despite remaining monophyletic in the phylogeny. Viruses in some of these individuals had a maximum genetic divergence approaching that found between two random, unrelated HIV-1 subtype-B pol sequences within the US population. Individuals with these highly divergent viruses tended to be diagnosed nearly a decade earlier in the epidemic than people with superinfection or virus with less intra-host genetic variation, and they had distinct transmission risk factor profiles. To better understand this genetic variation in cases with extremely divergent, monophyletic viruses, we performed molecular clock phylogenetic analysis. Our findings suggest that, like Hepatitis C virus, extremely divergent HIV lineages can be maintained within an individual and reemerge over a period of years.Understanding genetic variation in human immunodeficiency virus (HIV) is clinically and immunologically important for patient treatment and vaccine development. We investigated the longitudinal intra-host genetic variation of HIV in over 3,000 individuals in the US National HIV Surveillance System with at least four reported HIV-1 polymerase ( Within ons/site . In Nortons/site . Within e sweeps .HIV-1 superinfection occurs when an individual is sequentially infected with HIV from two different sources , which apol, with the intent of characterizing cases of superinfection in a US National HIV Surveillance System (NHSS). We employed a combined phylogenetic and genetic distance-based approach. As part of this investigation, we discovered a group of individuals with extremely divergent viral genotypes that were monophyletic in an HIV phylogeny. This finding suggests that extremely divergent HIV pol lineages can be maintained over the course of prolonged infection. Here, we characterize this unexpected pattern of HIV genetic variation and discuss implications for the detection of HIV molecular transmission clusters in a surveillance context.We investigated the longitudinal intra-host genetic variation of HIV pol sequences reported to the US NHSS from 2000 through Fall 2015 were included in the study  were excluded from subsequent analyses given the variable substitution rates across HIV subtypes (https://hivdb.stanford.edu/page/webservice/)  . Non-B ssubtypes . Drug reervice/) .pol sequences were aligned in pairwise fashion to a reference sequence . TN93  . Briefly9). TN93  genetic 9). TN93 . In an H9). TN93 . TherefoA maximum-likelihood phylogenetic tree was inferred from the 17,688 sequences using FastTree2 under a GTR + CAT20 model . Our incn = 2, 914 individuals], (2) monophyletic viruses with at least one consecutive strain exceeding 0.015 substitutions/site divergence [n = 240 individuals], and (3) polyphyletic viruses with at least one consecutive strains exceeding 0.015 substitutions/site divergence [n = 149 individuals]. We excluded individuals with monophyletic virus in which a single virus was >0.015 substitutions/site from all other viruses in that person, because these instances cannot be easily distinguished from poor sequence quality . We also excluded individuals with non-monophyletic virus where the maximum genetic distance was <0.015 substitutions/site, because these instances cannot be easily distinguished from transmission within a local transmission cluster or poor resolution in a large phylogenetic tree . Our final dataset comprised 3,303 individuals.Based on the genetic distance and phylogenetic analysis, we identified three populations for analysis: (1) monophyletic viruses with no consecutive strains exceeding 0.015 substitutions/site divergence [We performed multivariate multinomial logistic regression analysis to investigate differences in these three populations. This regression analysis included year of diagnosis; transmission risk factor ; and presence of common DRAMs . MSM who reported injection drug use were classified as PWID. Regarding DRAMs, we considered mixed populations (i.e. sequence ambiguities indicating the presence of both DRAM and wild-type variants) to be presence of a DRAM.−3 substitutions/site/year and standard deviation of 1 × 10−6. This calibration comes from previous molecular dating using NHSS data  in RDP4 we scanned for genetic recombination in 134 sequences from the 11 individuals with monophyletic viruses with the greatest intra-host genetic divergence .We interrogated the NHSS for evidence of superinfection. We identified instances in which virus from within a single individual was polyphyletic in the phylogeny and had a consecutively sampled virus that was >0.015 substitutions/site divergent. Of the 3,303 individuals infected with pure subtype B strains, 149 (4.5%) met these criteria for defining superinfection. Of these 149 individuals, only 9 individuals had viruses in which the divergent virus was genetically similar (≤0.015 substitutions/site) to another virus in the same host.To investigate patterns of longitudinal viral divergence, we identified a group for whom there was no evidence of superinfection: individuals with monophyletic virus in which consecutive viruses are never more than 0.015 substitutions/site divergent from the previous virus. Of the 3, 303 individuals infected with pure subtype B strains, we found 2,914 individuals (88.2%) who met these criteria. Unexpectedly, we found 240 individuals (7.3%) with monophyletic virus in which one or more consecutively sampled viruses was >0.015 substitutions/site from the previously sampled virus.Those 240 individuals who had highly divergent consecutively sampled viruses are similar to the extreme of the other 2,988 individuals with monophyletic virus (gray and red bars in The phylogenetic and genetic distance approach to characterizing superinfection is limited by the inherent difficulty in distinguishing within-host diversity from superinfection from another person with a closely related virus (i.e. superinfection from within a transmission cluster). Therefore, it is possible that the tail of the distribution of uppermost genetic distances for individuals with monophyletic virus is actually superinfection from a closely related source.P < 0.001; P = 0.024) or PWID  than individuals with extremely divergent consecutive viruses that were monophyletic (P < 0.001). DRAMs were significantly more common in individuals with monophyletic virus with extremely divergent, consecutively sampled virus than in individuals with monophyletic virus without extremely divergent virus  than individuals with either polyphyletic virus  or monophyletic virus with no extremely divergent strains  . We restricted this analysis to the eleven individuals with at least ten viral genotype sequences to more clearly understand patterns of viral genetic variation. Genotype sampling in these eleven individuals was dense over the observation period. The 3,303 individuals previously analyzed had a mean of 1.3 genotypes reported per person-year. Within these eleven individuals (denoted here as Cases A through K), there was an average of 2.2 viral genotypes per person-year  included the year of diagnosis . In threpol region in only one of these individuals  . We note Case I; . Nonethepol genotypes within the US NHSS. We found 149 (4.2%) individuals infected with highly divergent (i.e. >0.015 substitutions/site), consecutively sampled viral genotypes that were polyphyletic in a large HIV phylogeny. Surprisingly, we found >1.6-times as many individuals  with highly divergent, consecutively genotyped viruses that were monophyletic in the HIV phylogeny. This latter group was distinct from individuals with probable superinfection, comprising people who were diagnosed, on average, nearly a decade earlier than inferred cases of superinfection. Furthermore, the maximum genetic distance within these individuals with extremely divergent, monophyletic virus closely resembled the maximum genetic distance observed in individuals without evidence of superinfection or highly divergent, consecutively sampled viral genotypes.We report the results of an investigation into longitudinal genetic variation in HIV pol, of about 1 × 10−3 substitutions/site/year. However, this substitution rate is consistent with among-host evolutionary rates, and the intra-host substitution may be substantially faster  was assumed to be an instance of superinfection. Importantly, the mono-infected individuals in the The maintenance of such highly genetically divergent strains, though common in chronic infection of another RNA virus, Hepatitis C virus, , has notportance . A comprWe must consider the possibility that, rather than these cases representing the maintenance of extremely divergent populations, they are actually the result of superinfection from within a close-knit transmission cluster. This possibility would indicate that superinfection from within a transmission cluster occurs far more frequently than superinfection from an unrelated strain. Less plausibly, this possibility suggests that superinfection from individuals with closely related strains occurs preferentially in individuals with significantly older diagnosis dates and preferentially occurs among people with heterosexual risk factors. Although it is likely that some fraction of the individuals with extremely divergent, monophyletic strains are actually the result of superinfection, the substantial differences in time since diagnosis the between these monophyletic and polyphyletic groups suggests a different mechanism behind their genetic variation profiles.de novo mutations after the re-introduction of ART, pre-existing viral variants encoding drug resistance emerge into dominance. These pre-existing variants may also possess the necessary compensatory mutations to offset the fitness deficit arising from drug resistance mutations (We note that in many of instances of oscillation between genetically diverged clades within monophyletically infected individuals in this study, these clades can be distinguished by the presence or absence of drug resistance at M184V in reverse transcriptase . Resurgeutations . During utations . TherefoConducting this study in a surveillance setting presents several limitations. Attribution of samples to a different individual or unintentional merging of two individuals within a surveillance database could artificially increase our estimates of superinfection. Moreover, poor quality sequencing of a single viral genotype could artificially increase intra-host diversity yet preserve monophyly. Furthermore, this investigation was limited to the analysis of bulk Sanger consensus sequences, which are routinely reported as part of HIV molecular surveillance in the US. The lack of population-level resolution inherent in consensus sequences prevents us from obtaining a clear picture of longitudinal intra-host diversity in these cases of interest. If similar cases of extremely divergent monophyletic viruses can be found in well-documented research cohorts, more in-depth investigations into the intra-host viral population dynamics will likely be possible.Public health agencies within and outside the US are increasingly incorporating molecular sequence analysis into their HIV surveillance activities to identify growing transmission clusters . The disSupplementary DataClick here for additional data file.


Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics.Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the  They belong to two families: the aviridae ,14 and tiviridae . These tic group  referredic group ). All vi ericina ,19, whil ericina ,20. Even0 kb and  ericina , very feStudies of algal viruses suggest that they may be species-specific or strain-specific to their host, but many different viruses can infect the same species ,22,23. AIn order to expand our knowledge of marine algal virus ecology we have conducted a one-year metabarcoding study of algal viral diversity and dynamics in the Northern Skagerrak, South Norway. We addressed the following questions: 1) Which algal viral OTUs  are dominating the Outer Oslofjorden and can they be linked to characterized algal viruses? 2) How is the algal virus community composition and relative abundance changing over the year? 3) How do the algal viruses co-occur with various co-existing protists? 4) Can co-occurrence analyses give new information about potential virus-algal host relationships?Sea-water samples from the Outer Oslofjorden were collected monthly at the OF2 monitoring station  between March 2010 and June 2011 except for July 2010, when sampling was not performed. The samplings were performed at the same time of the day each month. Niskin bottles (4 × 5 L) attached to a CTD  rosette were used to collect water samples from a depth of 1 m (subsurface) for virus and protist collection (20 L). The water for virus samples were first filtered through a 200 µm mesh sieve to remove zooplankton and then through a 0.45 µm pore-size, low-protein binding, Durapore membrane filters of 142 mm in diameter (Millipore) mounted in a steel tripod  by peristaltic pumping . Further, samples were concentrated  to a final volume of 50 mL using the QuixStand benchtop system with hollow 100,000 pore size fiber cartridges (NMWC) and stored in sterile falcon tubes. Then, 10 × 250 µL of the virus concentrate were transferred into sterile cryo-tubes, flash-frozen in liquid nitrogen, and stored at −80 °C until further processing. Water for protist samples were filtered as described previously .−1 of DNA, quantified by Nanodrop, was sent for sequencing. Library preparation and Lib-A unidirectional amplicon sequencing was performed on 20 µL of the DNA sample (11 ng µL−1) in a Roche 454 GS-FLX Titanium . A total of 203,229 viral reads were obtained.Viral DNA extraction, purification, and PCR amplifications of a part of the MCP gene were done as described in detail in , with thFor the protist samples, nucleotide extraction was performed as described in Gran-Stadniczeñko et al. . ShortlyViral and protist 454 reads were processed through the QIIME v.1.9.1 pipeline . AmplicoA total of 582 vOTUs were obtained based on 127,348 reads. Putative spurious data , were removed leaving a total of 313 vOTUs. Subsampling was then performed to the minimum read number of 1696 with the “rarefy” option in the Vegan package in R . A complMimiviridae and 16 Phycodnaviridae viruses, the closest hits in ViroBLAST, and the top hits from the NCBI non-redundant protein database [A first taxonomic assignation of the vOTU nucleotide sequences was done with BLASTn against the Viral GenBank database in ViroBLAST with default parameters . In addidatabase . The topdatabase .addfragment algorithm which takes shorter sequences and adds it to an alignment, keeping the gaps, and the relative position of the characters in the original alignment [gappyout setting [Mimiviridae and 16 Phycodnaviridae from [Alignment construction started with sequences longer than 200 aa using MAFFT L-ins-i ,47. The lignment . Ambiguo setting . This aldae from  was consdae from , implemedae from , and visdae from .SparCC [SpiecEasi [SparCC was run with default settings and with 500 bootstraps. Two-sided pseudo p-values were calculated for both datasets independently and edges with a p-value of >0.05 and a correlation score of <|0.5| were deleted. Protist-protist associations were also deleted, since we were primarily interested in the associations between viruses and protists. The visualization of the networks was done with Cytoscape v3.6.1 [Association networks were constructed to analyze the co-occurrence of algae and viruses. The dataset was prepared for network construction after first filtering OTUs not present in at least 2 of the samples. The dataset consisted of 342 pOTUs recently described in [SparCC  as implepiecEasi . SparCC e v3.6.1 .Phycodnaviridae and Mimiviridae taxonomic composition, vOTU richness, and relative abundance through the year by high-throughput amplicon sequencing (metabarcoding) of 15 samples taken monthly from the Outer Oslofjorden, Skagerrak, Southern Norway. The conserved MCP gene was amplified from all samples and resulted in 126,775 reads of 200–400 bp in length, representing 313 putative algae-infecting vOTUs after filtration, with a 97% similarity clustering, the removal of singletons, and translation into amino acids (aa). The vOTUs were named according to decreasing relative abundance before subsampling. After subsampling to the lowest number of reads per sample (1696 reads), to be able to compare samples, 243 vOTUs remained. None of the rarefaction curves for the 15 samples reached a plateau, indicating that the sequencing depth was not sufficient to capture the full diversity at Outer Oslofjorden (We assessed the ofjorden . The mosofjorden .Haptolina ericina and Haptolina. hirta) and four prasinophytes . A total of 59 vOTUs best matched uncultured environmental MCP viral sequences. The rest had a lower similarity to cultured or uncultured reference sequences, meaning they had not been found elsewhere by metabarcoding.Of the 313 vOTUs, 95 had a similarity ≥90% to reference sequences obtained by BLASTn against Viral Genbank on the ViroBLAST platform . Of thesPhycodnaviridae and Mimiviridae phylogenetic trees based on MCP amino acid vOTU sequences were constructed including all 313 vOTUs  with a bootstrap value of 84%. Three vOTUs  clustered with the environmental OTU/M0501 from Raunefjorden (Western Norway [Haptolina hirta (HhV-Of01) with high bootstrap values (>80%). Furthermore, vOTU 12 and 15 clustered with sequences from a virus infecting Ostreococcus tauri (OtV1 165). The most abundant Mimiviridae vOTUs were vOTU 2 and 4. The vOTU 4 clustered with Chrysochromulina ericina virus  with a high bootstrap support (95%), whereas vOTU 2 did not cluster with any known viral reference sequence. Furthermore, a new branch consisting of two vOTUs (vOTU 5 and 16) was obtained as a sister to the Mimiviridae family . They wn Norway ) and a ve family .Phycodnaviridae family was more diverse than the Mimiviridae in all samples , had negative correlations with two chlorophyte pOTUs that had a best match to Pycnoccocus provasolii. (pOTU 58) and Pyramimonas sp. (pOTU 209), together with two dinoflagellate pOTUs (Karenia papillonaceae (pOTU 1). The Mimiviridae vOTU 2, which did not cluster with a known reference sequence, had a positive correlation with a picobilizoan (heterotrophic picoplankton) and a negative correlation with an uncultured dinoflagellate. The Phycodnaviridae vOTU 3 only presented negative correlations with two ciliates, one diatom, and one chrysophyte. vOTU 4, which clustered close to the Chrysocromulina ericina virus CeV, did not show any significant correlation with a haptophyte pOTU, but showed positive co-occurrences with two diatoms and a choanoflagellate (heterotrophic opisthokont protist). Furthermore, the Phycodnaviridae vOTU 7, showed negative correlations with a MAST (uncultured heterotrophic stramenopile), and the haptophyte Emiliania huxleyi, as well as positive correlations to two uncultured alveolates and a choanoflagellate. The remaining Phycodnaviridae and Mimiviridae vOTUs did not show significant correlations with potential protist hosts.Co-occurrence network analyses were done to detect positive and negative protists-virus correlations between our protist  and virute pOTUs , Table 1The seasonal dynamics of protists at the Outer Oslofjorden OF2 sampling campaigns have been described in . Figure M. commoda, M. sp. clade-B-subarctic, M. pusilla, and Haptolina sp., the vOTUs occurrence occasionally overlapped with that of the hosts’ and peaked after a drop of the hosts’ abundances, whereas no overlap between abundances of Micromonas bravo and MpV1 was observed.Combining the distribution pattern of the most abundant vOTUs assigned to a cultured virus with a known host, with that of their putative protist host , Figure  shows a Phycodnaviridae or Mimiviridae families with high bootstrap support (>60%). A total 12% (36 vOTUs) had a best match with BLASTp to a cultured algal virus, whereas 70% (218 vOTUs) had 89% or a lower similarity to any available virus sequences. Furthermore, by phylogenetic placement, the majority of the vOTUs formed clades without any reference sequence. This is in accordance with previous studies, e.g., by Clerissi et al. [Phycodnaviridae but were in our study placed in the Mimiviridae clade: ABU23699, ABU23704 [Phycodnaviridae. Recent phylogeny based on Nuclear Cytoplasmic Large DNA Viruses (NCLDV) orthologous genes, however, showed that many of the large dsDNA algal viruses belong to the Mimiviridae . The taMicromonas pusilla virus (MpV1), Haptolina hirta virus (HhV-Of01), Ostreococcus spp. viruses , and Chrysochromulina ericina viruses . Members of the genera Micromonas and Haptolina are common phytoplankton species in the Oslofjorden [Micromonas pusilla was until recently the only described species of this genus, but was divided into several species by Simon et al. [Micromonas commoda was the most abundant Micromonas species in Outer Oslofjorden OF2 station, but M. pusilla, M. bravo, and M. sp. clade-B-subarctic were also detected [Micromonas species as their hosts. The picoplanktonic prasinophyte genus Ostreococcus has a wide distribution in temperate to tropical marine waters, but was not recorded by metabarcoding north of 60°N by Tragin and Vaulot [Ostreococcus tauri, strain OTH95 [Ostreococcus mediterraneus. Due to this incongruence, we classified vOTU 12 and 15 as Ostreococcus sp. virus  to cultured marine viruses infecting different protist taxa such as ofjorden ,34,58. Mn et al. . Micromoin OTH95 . With BLp. virus . Both O.The viral community at Oslofjorden showed a clear temporal variation, but not a recurring seasonal pattern as has been demonstrated in other studies . The laMicromonas commoda, recently separated from Micromonas pusilla [M. pusilla, which decreased in abundance from September to October 2010, at the same time as the vOTU 1 was gradually increasing in abundance. The MpV1-like vOTU 1 was the most abundant vOTU and dominated for eight months. Furthermore, vOTU 3 had an affinity to MpV1 and was among the most abundant vOTUs from March to June 2010 and increased in abundance when the potential host M. commoda decreased. In a previous study of the Micromonas pusilla virus (MpV) in the Skagerrak-Kattegat area, Sahlsten [Micromonas infecting virus MpV to persist in the water for a long period. Additionally, Zingone et el. [Micromonas were resistant to viral infection. A coexistence between virus and host during long periods is the most observed pattern in our study, as most of our vOTUs are present at several timepoints  that varied similarly through the year. These positive/negative correlations do not necessarily imply that the viruses are able to infect such diverse hosts, but that they may influence one host group which in turn affect the growth conditions for others. Alternatively, the protist groups show similar or opposite responses to other environmental factors than viruses.Finally, the temporal variation in abundance of co-occurring viruses and protists  explainsPhycodnaviridae or Mimiviridae based on their major capsid protein amino acid sequence. Phycodnaviridae showed both the highest richness and relative abundance. We obtained 313 vOTUs and a majority did not match with any cultured or environmental sequences available in gene databases (70% at a ≥90% similarity). A total of 12% of the vOTUs had an affiliation to a known cultured virus with either a putative prasinophyte or haptophyte algal host. These hosts have all been found in the Outer Oslofjorden. The viral dynamics show a clear temporal variation with some vOTUs persisting for several months, suggesting they co-exist with their hosts, whereas others were present at specific time points, suggesting a possible boom and bust relationship with their hosts. The co-occurrence analysis could not with any certainty reveal new host relationships, but suggests some relationships to be examined in future studies. The comparison between the relative abundance of viruses and of their potential hosts can give new insight into the virus-algal host dynamics and the ecological role of algal viruses. This study also provides a baseline for future studies of algal virus diversity and temporal dynamics. With more cultured and genetically characterized viruses, more of the viruses reported here may be connected to a host and contribute to a better understanding of the ecological importance of algal viruses and their distribution in space and time.All vOTUs could be classified to either


Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation. Prunus armeniaca) is one of the most popular fruits in central Europe, especially in Hungary, where it is not only consumed as a fresh fruit, but also serves as a raw material for jam and “Palinka” (a distilled spirit) production. Thanks to intensive breeding programs since 1950, many varieties have become available with improved characteristics for these specific purposes. In accordance with the usual routine . As. As35]. sequence , supportNew high-throughput sequencing-based methods can provide valuable information about the presence of different viral pathogens. Due to their speed and reliability, they proved to be a suitable alternative to labor- and time-intensive biological indexing ,53. In ain vitro cultured plant, trees under the isolator net and in an open field in the stock nurseries suggested that while infection with LChV-1 happened in an open field from the surroundings, CVA infection originated from the propagation material. However, to test this hypothesis, the presence of LChV-1 in the surrounding cherry and sour cherry plantations together with the presence of mealybugs or other possible vectors should be tested.sRNA NGS technology helped us to describe the unknown and rare presence of LChV-1, in apricot. The virus diagnostics of the Prunus species and species present in the natural vegetation, further investigation is necessary.In this work, CVA and LChV-1 were first described in Hungary. To get a more detailed picture of their prevalence and distribution in different cultivated