The NYS and NYT glycosylated viruses showed higher viral replication, and lower pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A’DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status.The N-linked glycosylation motif at amino acid position 154–156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A’DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for  Culex mosquitoes are the primary hosts for WNV natural transmission cycles. The envelope (E) protein for WNV contains a variable N-linked glycosylation motif which influences avian replication, mosquito infectivity and vector competence. WNV isolates with variable E protein glycosylation motifs that have been historically associated with human cases of disease, were selected to generate WNV glycosylation mutant viruses via reverse genetics. Replication capacity and host competence of WNV glycosylation mutant viruses were compared in vitro and in vivo in American crows, house sparrows and Culex mosquitoes. The data demonstrated that N-linked glycosylation was not as crucial for WNV transmission and host competence as previously reported. Rather the amino acid identities of the glycosylation motif were more important in dictating WNV virulence phenotypes in both avian and mosquito host.West Nile virus (WNV) has been responsible for the largest human encephalitis epidemics in the continental United States. Avians and  First isolated in 1937 from a febrile patient in the West Nile district of northern Uganda  7] and AYS to IYS, NYE or YS to IYS, were reYS to IYS, 37, manhenotypes, 40, indUnlike previous studies in which the growth of WNV mutants in AMCR monocytes predicted viremia potential and virulence, all virCx. quinquefasciatus. Even after bypassing the midgut barrier of infection by intrathoracic inoculation, NYP produced significantly lower viral titers in the saliva. The attenuated profile of NYP virus in Culex spp. in the present study could explain the low frequency of isolation of WNV-NYP in nature[Cx. tarsalis could preclude subsequent transmission. In contrast to our study, Murata et al reported the isolation and characterization of a small plaque variant with the NYP motif where no oral infectivity differences were observed with Culex pipiens pallens and disseminated viral titers were indistinguishable between the NYS and NYP viruses[Culex spp. and more efficient transmission rates at warmer temperatures[The specific amino acid identity at the E-156 locus influenced mosquito oral infectivity independent of glycosylation status as the NYT virus had a higher infection rate than NYS. The NYP mutant exhibited a severely debilitated infection and dissemination phenotype while the NYF did not significantly affect these phenotypes compared to the NYS virus. The E-154 locus showed similar variable effects on vector competence with restricted infectivity of the KYS mutant observed when compared to the SYS mutant. With the exception of the NYP mutant virus, absence of E protein glycosylation did not significantly attenuate viral growth, infection and/or dissemination rates of NYF, SYS, SYP and KYS viruses when compared with NYS parental WNV in in nature, 21. Whiin nature in highlP viruses. AlternaP viruses, 43. Thiperatures. This E-in vitro studies with recombinant rabies virus glycoproteins have shown that differential glycosylation efficiencies of the N-linked moieties were dependent upon the hydroxyl amino acid in the glycosylation motif[Interestingly, the midgut infection rate for NYT 70%) was more than twice that of the NYS virus (34%), indicating that a single amino acid polymorphism at E-156 that maintained glycosylation status of the E protein could result in a significant effect on vector competence. Nevertheless, the role of glycosylation for modulation of this phenotype between these viruses cannot be discounted as ion motif. That stion motif, 46. Thu0% was moion motif.Culex mosquitoes when compared with WNV[Cx quinquefasciatus. The effect of the different E154-156 mutations on avian viremia potential was variable and species-specific, indicating that different mutations could be competent for avian viremia generation in certain avian species. Despite having delayed viremia onset, even the A’DEL mutant was capable of eliciting avian viremia conducive for mosquito infection, although this mutant failed to demonstrate transmissibility in Cx quinquefasciatus. Data presented herein indicates that WNV E154-156 motif amino identity, rather than specific N-linked glycosylation, dictates acute viral titers in birds and vector competence in mosquitoes and the effect of a particular E154-156 moiety expressed on WNV avian and vector competence was species-dependent. The isolation of many of these E-154-156 mutants in the field is intriguing and coupled with the findings here indicates the strong possibility that non-glycosylated WNV variants could circulate at relatively high frequency. The frequency of WNV genotypes to express a particular E-glycosylation motif (E154-E156) is likely due to selection pressures on viral replicative fitness by the dominant amplification host(s) and vectors utilized during local transmission events. Future studies should be designed to assess the potential fitness advantages for these variants in specific enzootic vectors and/or hosts.Other flaviviruses such as Yellow fever virus and St. Louis encephalitis viruses do not encode a glycosylated E protein, 47, 48. with WNV, again d with WNV, 50 and


Variation in disease incidence in wildlife is often assumed to reflect environmental or demographic changes acting on an endemic pathogen. However, apparent endemicity might instead arise from spatial processes that are challenging to identify from traditional data sources including time series and field studies. Here, we analysed longitudinal sequence data collected from rabies virus outbreaks over 14 years in Costa Rica, a Central American country that has recorded continuous vampire bat-transmitted rabies outbreaks in humans and livestock since 1985. We identified five phylogenetically distinct lineages which shared most recent common ancestors with viruses from North and South America. Bayesian phylogeographic reconstructions supported bidirectional viral dispersals involving countries to the north and south of Costa Rica at different time points. Within Costa Rica, viruses showed little contemporaneous spatial overlap and no lineage was detected across all years of surveillance. Statistical models suggested that lineage disappearances were more likely to be explained by viral extinctions than undetected viral circulation. Our results highlight the importance of international viral dispersal for shaping the burden of rabies in Costa Rica, suggest a Central American corridor of rabies virus invasions between continents, and show that apparent disease endemicity may arise through recurrent pathogen extinctions and reinvasions which can be readily detected in relatively small datasets by joining phylodynamic and modelling approaches. Underst strains ,11. ConsDesmodus rotundus) rabies virus (VBRV) exemplifies a zoonosis for which limited understanding of transmission dynamics in a wildlife reservoir poses substantial management challenges. Although VBRV was discovered over a century ago, it remains the primary cause of uniformly lethal human and animal rabies outbreaks throughout most of the Latin American tropics and subtropics [Vampire bat  and South America  that sustain high incidence of human and/or livestock rabies –19. ThesLittle is known of the ecology of VBRV circulation in Central America, although rising human rabies incidence has been noted ,25,26. H2.(a)KU550098) was also from a vampire bat-associated lineage [r = 0.70, p = 0.006).Over the time period of this study (2004–2017), 60 rabies outbreaks were reported in domestic animals from Costa Rica, involving 180 mortalities . All animals were reported by their owners to have clinical signs consistent with rabies encephalitis, and brain samples were confirmed rabies positive by direct immunofluorescence by the National Veterinary Service Laboratory (LANASEVE). We sampled representative viruses collected from 39 cattle and 1 domestic dog from 38 outbreaks. Importantly, as cattle do not transmit rabies intra-specifically or back to bats, each record represents an independent spillover from the bat reservoir. The single rabies virus from a domestic dog (GenBank accession no.  lineage . The numhttp://rabv.glue.cvr.ac.uk), which maximized our ability to detect incursions into or out of Costa Rica [erminator v. 3.1 using the following cycle conditions: 30 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min; products were purified with BigDye XTerminator. DNA sequencing was performed on a 3130 Genetic Analyzer (Applied Biosystems). Sequences were aligned using MAFFT and trimmed to the coding regions [Upon arrival to LANASEVE, samples were stored at −80°C. RNA extractions used the DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer's instructions. Reverse transcription–polymerase chain reaction (RT–PCR) used the primers RAB20 5′ ACGCTTAACAACAARATCARAG-3′ and RAB304 5′-TTGACGAAGATCTTGCTCAT-3′ targeting the complete nucleoprotein gene . The nucsta Rica ,29. One-(b)n = 75) and without  sequences from other North, Central and South American countries. International analyses focused on the time scale and geographical patterns of viral dispersals into and out of Costa Rica using discrete phylogeographic analyses. We used a cut-off of 98% similarity to any Costa Rican sequence to identify VBRVs available in GenBank that could plausibly have shared a most recent common ancestor (MRCA) with Costa Rican viruses, but also included additional representative VBRV lineages for reference. National data were used to confirm the geographical origins suggested in the international analyses and to examine patterns of viral diffusion within Costa Rica using continuous phylogeographic analyses.Analyses used two datasets comprising sequences from Costa Rica with . Preliminary phylogenetic analyses of the international dataset in BEAST v. 1.8.4 used stepping stone sampling to estimate the marginal likelihood and Bayes factor (BF) support for the strict, relaxed lognormal and random local clock models  [−4, s.d. = 2 × 10−4). Molecular clock models were not formally compared for the national dataset since the random local clock cannot currently be applied to single analyses with multiple trees (see below). We used a relaxed lognormal clock (uniform prior from 1 × 10−4 to 7 × 10−4) since substantial rate variation among branches (ucld.stdev > 0.65) rejected the strict molecular clock. We used partitionFinder2 to identify the best fitting models of codon partitioning and nucleotide substitution, according to the Akaike information criterion corrected for small sample size [Both datasets had evidence of clock-like evolution according to TempEst  ,32. The ple size . Internan = 13 and 14 sequences, respectively) as separate data partitions in the same BEAST analysis [Seraphim package in R [racer v. 1.7.1 to verify parameter convergence, check that effective sample sizes on parameters exceeded 200 and select appropriate burn-ins.Discrete phylogeographic modelling in BEAST used country of viral origin as a categorial trait and was set to infer asymmetric rates between countries . Models analysis . Specifiage in R ,38. We m(c)multinom function in the nnet package of R. Specifically, we modelled the identity of the viral lineage associated with each rabies outbreak as a nominal dependent variable with five levels (the number of discrete virus lineages detected), with year as the only covariate. This analysis used the earliest detected virus (L2) as a baseline outcome against which to compare other lineages (i), leading tob,i0 is the intercept for virus i and i,bL2 is log odds effect of the change in year on an outbreak being caused by virus i versus virus L2. Results were qualitatively similar for all alternative baselines. We calculated a pseudo-r2 and p-value for the effect of year by comparing the log likelihood of the fitted model against an intercept-only model using the pscl package. Second, we used the number of months between successive detections of the same viral lineage  to assess evidence for continued viral circulation at subdetectable levels versus extinction. We compared the intervals between successive detections of each virus while it was known to have circulated  to the interval between the final detection of each virus and the end of the observation period, capped by the last outbreak in our dataset (2017.66). Values for the time since final detection are therefore conservative lower bounds, representing minimum estimates. The empirical probability density of inter-detection intervals was estimated using the demp function in the EnvStats package of R, after aggregating intervals observed across all viruses. To avoid artificially reducing the distribution of inter-detection intervals, we removed repeat observations of the same virus in the same month . The pemp function was used to estimate the probability of virus lineage extinction, given the number of months since its final detection. R scripts for both statistical analyses are provided in electronic supplementary material, file S3.We evaluated evidence for viral invasions and extinctions given the possibility of undetected viral circulation using two distinct approaches. First, we tested whether the probability of observing each viral lineage varied through time by fitting a multinomial logistic regression using the 3.a). Across the entire tree, the VBRV nucleoprotein evolved at a median rate of 4.86 × 10−4 substitutions per site per year (95% highest posterior density (HPD) = 3.54–6.34 × 10−4), which was similar to previous estimates [a). Consequently, the discrete phylogeographic analysis identified repeated bidirectional viral dispersal between Costa Rica and Colombia (b). This analysis further supported a dispersal of an L1 virus from Costa Rica to El Salvador, consistent with the spread of South American viruses to more northern Central American countries via Costa Rica. Lineage 2 (L2) was detected between 2004 and 2014 and shared an MRCA with Mexican viruses in 1999.8 (95% HPD = 1995.3–2003). The phylogeographic analysis supported unidirectional viral dispersal from Mexico to Costa Rica, suggesting the southward invasion of a North American virus (b).Phylogenetic analysis of the international dataset revealed two main paraphyletic VBRV lineages that circulated in Costa Rica between 2004 and 2017 a. Acrossstimates . LineageColombia b. This aan virus b.Figure−1 (95% credible region on the mean branch velocity = 4.08–59.93) while in Costa Rica. The inferred ancestor of virus L2 occurred further north in central Costa Rica, but was considerably less precise, suggesting that the longer time period of circulation in Costa Rica largely erased evolutionary signal of viral origins .Ancestral state reconstructions of the latitude and longitude of the progenitors of the L1a and L2 viruses  provided additional support for invasions suggested by the discrete phylogeographic analysis. The inferred ancestor of virus L1a occurred in southern Costa Rica near the border with Panama, consistent with a recent invasion from South America . This in origins . This leb). Indeed, during the final 2 years of observation (2016–2017), only L1a viruses were encountered and cases occurred exclusively in the south of the country. Despite the relatively small sample sizes underlying the lineage-specific time series, the multinomial regression found an effect of year on which virus was observed  and had sufficient power to detect viral invasions and extinctions, evidenced by 95% CIs on the predicted probability of viral presence through time rising above or approaching zero, respectively (a). L1a and L1c were unlikely to have circulated in Costa Rica during the first 6 years of the study (mean probability of presence less than 0.01) and viruses L1d, L1b and L2 had a high probability (0.975–0.999) of having gone extinct by 2017 (a). Rabies outbreaks from 2018 (the year after our sequencing completed) revealed no additional cases in other regions, suggesting the continued absence of other viral lineages . By the end of the molecular study, non-L1a viruses had not been encountered for 2.96 years on average (range: 2–10.41 years), which was longer than the expected interval between successive detections of persisting viruses (b). The estimated probabilities of failing to detect a still-circulating virus were less than 0.005 for viruses L1b, L1d and L2, and 0.11 for virus L1c, confirming that all viruses other than L1a were likely to have gone extinct from Costa Rica. By combining molecular clock estimates of the time since the MRCA of clades with the final dates of viral detections, we estimated viruses circulated in Costa Rica for only 10.5 years on average (range: 4.7–18.65 years).Within Costa Rica, viruses showed variable degrees of temporal residence and spatial structure, and no virus lineage was detected across the duration of the study . For exafigure 3ectively a. L1a an by 2017 a. Rabies viruses b. The es4.Distinguishing endemic pathogen circulation from extinction–recolonization dynamics is a major challenge which has potential to undermine strategies for disease prevention and control. Focusing on Costa Rica, a small Central American country that has detected rabies nearly every year for decades, we observed invasions of North and South American viruses as well as northbound and southbound exportations of VBRV; however, all viruses had relatively brief residence times within the country. These observations are most parsimoniously explained by a dynamic process of spatial invasion fronts among Central American countries, each of which has limited capacity to sustain enzootic transmission in isolation. Consequently, patterns of spillover to humans and livestock reflect epidemiological processes occurring across multiple countries, which creates challenges and opportunities for prevention and control.Apparent viral extinctions and reinvasions might alternatively arise from cryptic, enzootic circulation of viral lineages, with periodic detection driven by fluctuating incidence in bat populations or inconsistent surveillance effort through time. Indeed, a variety of factors have been suggested to alter VBRV incidence in bats or spillover to humans or livestock, including temperature and precipitation associated with El Niño oscillations, changes in prey availability or quality, and age-structured transmission ,39–41. HLocally sporadic viral presence also helps explain why bat culls have not reduced VBRV spillover ,44. Sinc−1) and elsewhere (16.1–37.1 km yr−1 in Peru and Argentina) made detection of the same lineage in both continents unlikely during our observational window [b). This raises the possibility that the reliance of VBRV on sufficiently sized bat metapopulations for long-term maintenance combined with the narrow geography of Central America increases the probability of viral extinction [Our findings also illustrate a Central American corridor of VBRV between North and South America. Inter-continental spread of VBRV was previously implied by large-scale phylogenetic evidence that viral variants occur on both continents, but whether this reflected ancient or modern spread was unknown . The recl window ,18,24. Itinction . Consisttinction . Ultimattinction .R0 of rabies in bats, which creates frequent stochastic extinctions that leave large fractions bat populations susceptible for later viral invasions [The pattern of recurrent lineage invasion and replacement we observed provides new insights into the epidemiological mechanisms that underlie rabies endemicity. The ability of successive viral introductions to invade despite the recent presence of other viruses is unlikely to be explained by antigenic differences that allow viruses to evade host immunity as rabies antibodies are broadly protective within and among divergent lyssavirus species . Rapid rnvasions . The lownvasions . Howevernvasions ,51. ThesIn conclusion, this study shows that combining phylogenetic inference of relatively small amounts of longitudinal pathogen sequence data with statistical analyses of surveillance data can disentangle endemic pathogen maintenance from recurrent epidemic fadeouts and spatial re-introductions. For VBRV, this information reveals previously unrecognized spatial structure, clarifies the mechanisms underlying ecological or environmental drivers of cross-species transmission and provides new opportunities for prevention and control. The increasing availability of molecular sequence data should make similar approaches practical in other wildlife disease systems where pathogens persist through spatio-temporal processes, but the dynamics of spatial spread are unobservable from ecological data.


Cryptococcus gattii sensu lato isolations occurring during 1989–2016. Each system suggests the fungus was isolated in temperate climates before the 1999 outbreak on Vancouver Island, British Columbia, Canada. However, the Köppen-Geiger system is more precise and should be used to define climates where pathogens are detected.We compared 2 climate classification systems describing georeferenced environmental  Cryptococcus gattii sensu lato on Vancouver Island  to map global distribution. Geographically defined human and animal records of C. gattii infection are not always accessible because of privacy restrictions. In addition, the dates and locations of C. gattii s.l. exposures are often uncertain because of the mobility of animals and humans and C. gattii’s long, undetermined incubation and latency periods , tropical, subtropical, and temperate regions are denoted by latitudinal boundaries (http://koeppen-geiger.vu-wien.ac.at/shifts.htm). Using ArcMap version 10.5.1 , we overlaid the isolation coordinates of C. gattii on each map and extracted the corresponding climate classifications. If exact coordinates of the sample were not specified, we used ArcMap to estimate coordinates for the centroid of the park, city, or town where the sampling occurred. We first compared the climates assigned to isolations by classification system and then determined which positive sampling years had >1 isolation in a temperate region and how many of these years preceded 1999.According to the solar definition (the predominant definition used in the C. gattii s.l. isolations as tropical, subtropical, or temperate (>1 temperate-climate environmental C. gattii isolation (The solar definition classified the 83 environmental emperate . By compemperate . Both sysolation .C. gattii s.l. isolated on Vancouver Island during the outbreak as temperate (C. gattii s.l. isolation coordinates from only 1 year before 1999 could be classified as temperate (Both variations of the solar definition and the Köppen-Geiger system classified the environmental samples of emperate  Table. Aemperate . By contemperate  Table. Temperate  Table, bC. gattii s.l. However, in terms of geographic scale, the solar definition of tropical, subtropical, and temperate are too coarse for the purposes of classifying or describing areas of local clinical, veterinary, or environmental isolations of C. gattii s.l. or any other environmentally contracted pathogen. Species, including pathogenic species, can live within geographically smaller refugia that maintain their climatic and biological needs across larger landscapes and solar boundaries, depending on topography, microclimates, and habitat fragmentation (Both versions of the solar definition and the Köppen-Geiger system suggest that the Vancouver Island outbreak was not the first-ever temperate detection of C. gattii samples in the English language peer-reviewed literature. As a result, our findings are an underrepresentation of the full global extent of C. gattii s.l. in the environment. Other evidence exists for the emergence of C. gattii s.l. in temperate climates before 1999. For example, in addition to the environmental isolations made in Busselton, Western Australia, Australia (C. gattii infections in animals were reported in southwestern Australia, including Perth, before 1999 (C. gattii sampling, and others provided a park or city name. Providing the exact coordinates offers the greatest certainty of a detection location and better precision in climate classification.One limitation of our study was dependence on the reporting of environmental ore 1999 . AnotherC. gattii s.l. as an example for mapping georeferenced pathogen isolations worldwide, we demonstrated the opportunity to improve pathogen monitoring through the development of a standardized global climate classification framework. Using more spatially specific climate classification methods, such as the Köppen-Geiger system used by medical geographers, coupled with the continued reporting of pathogen isolation locations, will improve comparability of pathogen detection in new natural environments.By using Cryptococcus gattii sensu lato using a climate classification system–based approach.Additional information for determining the temperate climate detection of


Aedes aegypti larvae hatched from eggs collected in Jojutla, Morelos, Mexico. Of the 151 pools analyzed, 17 tested positive for Zika virus RNA; infectious Zika virus was successfully isolated from 1 of the larvae pools (31N) in C6/36 cells. Real-time quantitative PCR and indirect immunofluorescence assays confirmed the identity of the isolate, named Zika virus isolate 31N; plaque assays in Vero cells demonstrated the isolate’s infectivity in a mammalian cell line. We obtained the complete genome of Zika virus isolate 31N by next-generation sequencing and identified 3 single-nucleotide variants specific to Zika virus isolate 31N using the meta-CATS tool. These results demonstrate the occurrence of natural vertical transmission of Zika virus in wild Ae. aegypti mosquitoes and suggest that this transmission mode could aid in the spread and maintenance of Zika virus in nature.We characterized natural vertical transmission of Zika virus in pools of  Flavivirus, family Flaviviridae. Zika virus is closely related to other viruses of medical importance, such as dengue, West Nile, and yellow fever viruses  that is classified in the genus Aedes mosquito species  during interepidemic periods is thought to be caused by vertical transmission of the virus from the infected adult mosquitoes to their offspring for Ae. aegypti mosquitoes has been evaluated under laboratory conditions by searching for the presence of Zika virus RNA in several organs from the offspring of infected mosquitoes, demonstrating the presence of viral RNA in the guts and salivary glands of the offspring .In this study, we sought to demonstrate natural vertical transmission in We collected mosquito egg samples in the municipality of Jojutla, located in the southern region of the state of Morelos, Mexico . Arboviral infections are common in this municipality, which is usually hot and dry during October–April and warm and humid during May–September. During 2016, the incidence of Zika virus infections increased in Morelos; 269 cases were reported by the end of the year, of which 69 cases were from Jojutla. The reported cases in Jojutla were recorded as follows: 1 in August, 17 in September, 20 in October, 25 in November, and 6 in December , following regulations for entomological surveillance with ovitraps (NOM-032-SSA2–2014). In 2016, 180 ovitraps were placed and maintained throughout the year in different locations in Jojutla (We collected  Jojutla . We dete3 in Hank’s solution) using a pestle mixer  and stored pools at −80°C for further analysis.We placed the egg papers in water containers and incubated them at 37°C for 1 wk under 24-h light/dark cycles. After the incubation period, we collected larvae in stages 2–3 and separated them into pools of 20–30 larvae. We macerated each of the pools in viral transport media  for 10 min, extracted total RNA from the lysates using Zymo-Spin RNA extraction columns , and quantified the RNA with a Nano Drop 2000 . We detected the presence of Zika virus RNA by quantitative reverse transcription PCR (qRT-PCR) using the TaqMan system (https://www.thermofisher.com) with primers Zikv 1086 Fw 5′-CCGCTGCCCAACACAAG-3′ and Zikv 1162c Rv 5′-CCACTAACGTTCTTTTGCAGACAT-3′; and probe Zikv 1107-FAM probe 5′-AGCCTACCTTGACAAGCAGTCAGACACTCAA-3′, as previously described by Lanciotti et al. . Twenty-four hours before infection, we seeded C6/36 cells in 24-well plates at 80% of confluence in growth medium  and incubated them at 28°C in a 5% CO2 atmosphere. Cells were adsorbed with the clarified larvae macerates for 2 h at 28°C. After the incubation period, we added fresh maintenance medium to the cells and left the infection to proceed for 7 d at 28°C and a 5% CO2 atmosphere until a cytopathic effect was observable. We tested the supernatants of the infected cells for the presence of Zika virus RNA by qRT-PCR, as described earlier in this section. We performed 4 passages of each isolate in C6/36 cells to increase viral titers for NGS.We selected the larvae pools that tested positive for Zika virus RNA and displayed the lowest quantitation cycle (Cq) values (range 22.3–33.4) for viral isolation. In brief, we diluted macerated larvae pools in maintenance medium  and filtered them through 0.22 μm membranes  for 1 h. We washed the cells twice with PBS and then mounted them with VECTASHIELD medium with DAPI  for confocal microscopy .For the indirect immunofluorescence assays, we grew Vero cells over glass coverslips and then infected them with the Zika virus isolate. At 48 hours postinfection, we fixed cells with 4% paraformaldehyde and then incubated them with ice-cold methanol for 10 min. We blocked the cells using 10% FBS in phosphate-buffered saline (PBS) for 1 h and then incubated them overnight at 4°C with a 1:500 dilution of the monoclonal antibody 4G2. After the incubation period, we washed the cells twice with PBS and then incubated them with an Alexa Fluor 488-conjugated goat antimouse IgG antibody , 2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine  and incubated at 37°C for 4 d. We fixed the cells with 4% paraformaldehyde in PBS and counterstained them with crystal violet-formaldehyde (Sigma-Aldrich).For the plaque assays, we seeded Vero cells in 24-well plates until they reached a confluence of 80%. We used 10-fold serial dilutions of the Zika virus isolate to infect the cell monolayers and left them to adsorb for 1 h. After the adsorption period, we removed the virus and overlaid cell monolayers with 1 mL of DMEM (Invitrogen) supplemented with 2% carboxymethyl-cellulose . We constructed RNaseq Illumina shotgun libraries at the Unidad Universitaria de Secuenciación Masiva y Bioinformática-Instituto de Biotecnología and sequenced them using paired-end sequencing with a MySeq system . We assessed the quality of reads using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc); low-quality positions and reads were eliminated using in-house scripts. We assembled the valid reads without reference using the program Trinity 2.8 (http://blast.ncbi.nlm.nih.gov/Blast.cgi).For the complete genome sequencing of the Zika virus isolate, we depleted 200 ng of total RNA extracted from the supernatants of infected Vero cell monolayers of rRNA using the NEBNext rRNA Depletion Kit (human/mouse/rat) following the manufacturer’s instructions (New England Biolabswww.viprbrc.org). We removed identical sequences and those with undetermined bases from alignment. We applied near-identity clustering (0.999) to the remaining sequences in Cd-hit-test  RNA sequences from the Virus Pathogen Resource (ViPR) database  of the pools tested positive: 9 (5.7%) from the larvae raised from the eggs collected in June and 8 (5.1%) from larvae raised from the eggs collected in November. To determine the proportion of infected larvae in the population, we calculated MIRs for the 2 collection periods (June and November 2016) and found an increase in the MIR observed from the larvae raised from the eggs collected in June (2.5) to the MIR from the eggs collected in November (6.9) .To confirm the presence of infectious Zika virus in the larvae pools, we used 11 of the 17 pools that tested positive for Zika virus RNA that displayed the lowest Cq values to attempt viral isolation. We were able to isolate infectious Zika virus from only 1 of the larvae pools (31N), as determined by the presence of cytopathic effect in C6/36 cells characterized by monolayer detachment , panel AThe ability of the 31N isolate to infect a mammalian cell line was confirmed by the appearance of the characteristic cytopathic effect of Zika virus in infected Vero cell cultures, characterized by cell rounding and detachment , panel AAedes mosquitoes obtained in the state of Chiapas, Mexico, had the highest identity (98.8%) with our isolate.We isolated viral RNA from the supernatant of Vero cells infected with the fourth passage of Zika virus isolate 31N and processed it by RNA sequencing by the NGS MiSeq Illumina protocol. Around 40% of the total reads  were assembled in a single contig of 10,795 nt in length, with a mean depth of 42,000 reads. BLAST results of the assembled contig revealed that the strain ZIKV/Aedes.sp/MEX/MEX_I-7/2016, which belongs to a Zika virus isolated from We identified single-nucleotide variants (SNVs) in the Zika virus isolate 31N genome by aligning the sequence with other mosquito- and human-derived sequences obtained from the ViPR database and running the meta-CATS analytic tool, as described in the Materials and Methods section. To carry out this analysis, we grouped the sequences by the host of origin. We identified SNVs in positions 3176, 3286, and 5636 that were unique to the larva genome. On the other hand, the SNVs in positions 2071 and 3333 were found in human-derived sequences but were not present in other mosquito genomes. The SNV in position 2071 was shared only with a sequence from French Polynesia, likely an example of parallel evolution, whereas the polymorphism in position 3333 was common with 2 sequences from Mexico and may correspond to a local variant. Finally, we found 8 SNVs that were also present in other human and mosquito sequences but were more common in mosquitoes. Only 1 SNV was found in the E gene, whereas the rest were found in nonstructural (NS) genes: 5 in NS2A, 4 in NS3, 2 in NS5, and 1 in NS1 .To characterize the evolutionary relationship between the 31N isolate and other Zika virus genomes that have been previously reported, we performed a phylogenetic analysis. The phylogenetic reconstruction of the complete genomes of 98 Zika virus sequences, including the 31N isolate, revealed that the 31N isolate belongs to the Asian-American lineage of Zika virus and clusters together with other sequences of human and mosquito origin from Mexico .Natural vertical transmission of mosquito-carried viruses has been proposed as one of the main ecologic processes involved in the maintenance of these viruses in susceptible mosquito populations, particularly during interepidemic periods and harsh climate conditions when horizontal transmission becomes difficult  calculated from the larvae hatched from the eggs collected in November 2016, in contrast to the MIR from the larvae hatched from the eggs collected in June (2.6), might be correlated with the increased number of human Zika virus cases that were reported in Jojutla during November, whereas during June no human cases were reported. The absence of reported Zika virus human cases in June could be the result of asymptomatic cases, cases clinically misdiagnosed as dengue virus infections, or both.In this study, we demonstrated the occurrence of natural vertical transmission of Zika virus in wild mosquito populations from the municipality of Jojutla in the state of Morelos, Mexico. The RNA of Zika virus was detected in 17 larvae pools; the rates of vertical transmission of Zika virus in the wild The higher number of persons infected with Zika virus in Jojutla during November reflects an increase in the number of infected mosquitoes resulting from horizontal transmission of the virus between mosquitoes and infected humans. It is possible that vertical transmission is contributing to the number of infected mosquitoes, which are, in turn, capable of transmitting this virus to a higher number of humans. However, the relative importance of vertical transmission for the maintenance and spread of the virus cannot be elucidated with the data available so far.Ae. aegypti mosquitoes ranged from 4.9 to 24.6, depending on the strain of the virus tested  tested, which usually causes an underestimation of the real number of infected mosquitoes in a population (Ae. aegypti mosquitoes by the successful isolation of infectious Zika virus (31N) from larvae raised from field-collected eggs. This evidence strongly suggests that infectious Zika virus can be transmitted from adult female mosquitoes to their offspring and increases the evidence of the role of natural vertical transmission in the maintenance of Zika virus in wild mosquito populations.In this work, we were also able to demonstrate the natural vertical transmission of Zika virus in The isolation of infective Zika virus vertically transmitted from adult mosquitoes to their offspring suggests that this virus could be potentially transmissible between mosquitoes and their vertebrate hosts; nevertheless, this transmission still needs to be demonstrated. Moreover, we were able to detect several SNVs in the larvae-derived Zika virus isolate 31N, of which 3 were specific to this larvae-derived genome, as well as 10 others that were shared between the larvae and other mosquito and human sequences. Although it is plausible that these SNVs were acquired during virus culture, we think this is unlikely because 72 of the sequences included in the alignment corresponded to cultured viruses and none of them presented these mutations; however, the sequences from more larvae-derived viruses need to be determined to establish the significance of these SNVs. The larvae-specific SNVs were located in the coding regions for the NS2A and NS3 proteins, which are involved in the replication of the viral RNA (Ae. aegypti mosquitoes. Our results suggest that this transmission mode could aid in the spread and maintenance of Zika virus in nature, expanding the ongoing zoonotic threat from this virus to human health.In summary, we demonstrated natural vertical transmission of Zika virus in wild


Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been an important and widely used technique for discovery, identification and characterization of new viruses. Using TEM, viruses can be differentiated by their ultrastructure: shape, size, intracellular location and for some viruses, by the ultrastructural cytopathic effects and/or specific structures forming in the host cell during their replication. Ultrastructural characteristics are usually sufficient for the identification of a virus to the family level. In this review, we summarize 25 years of experience in identification of novel viruses from the collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for identification and characterization of new viruses. Two TEM techniques are generally used for this purpose: negative staining on an electron microscopic grid coated with a support film, or (ultra) thin-section TEM of infected cells, fixed, pelleted, dehydrated and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or on cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions  or on concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, so either high multiplicity of infection (MOI) or rapid virus multiplication is required.4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic, usually LR White .Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location, or from the ultrastructural cytopathic effects and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus to the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspensions before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which may not available in case of a novel virus. For on-section immuno-EM, OsOThe ultrastructure of most common viruses is well documented in good atlases and book chapters ,10,11,12The WRCEVA serves as a virus reference center for the world. Any zoonotic virus suspected of being biologically transmitted by arthropods or vertebrates is accepted for identification and characterization. A repository collection of more than 9000 virus strains is maintained with complementary sera and diagnostic antigens. In addition to arthropod-transmitted viruses, a number of other zoonotic vertebrate viruses  together with their respective antigens and antisera are also included in the reference collection. Many of the latter viruses were initially isolated and characterized by arbovirologists, as these agents have occasionally been isolated from clinical samples taken from wild animals or people during arbovirus field studies.The WRCEVA is a direct outgrowth of the worldwide network of laboratories, first established in the 1950s by the Rockefeller Foundation, to study the role of arthropod-borne viruses in producing human and animal disease and the mechanisms by which these viruses are maintained and transmitted in nature. When this program was initiated at the Rockefeller Foundation Virus Laboratories in New York City in 1951, fewer than 28 arboviruses had been described; and only a few, such as yellow fever, some of the encephalitides, and dengue were known to cause serious disease in human beings. Concurrent with the initiation of the Rockefeller program, the U.S. Army, Navy, Public Health Service and several foreign governments also established arbovirus laboratories and field research programs. This network of field laboratories relied on the Foundation’s central virus reference laboratory in New York until 1964, when the laboratory and some Rockefeller staff were moved to Yale University, and the Yale Arbovirus Research Unit (YARU) was established. In the mid-1995, Drs. Robert Tesh and Robert Shope, at that time directors of the WRCEVA, moved from YARU to the Center for Tropical Diseases  at the University of Texas Medical Branch (UTMB) in Galveston, and brought the reference collection with them. The decision to establish the Reference Center at UTMB was based in part on the willingness of the University of Texas to provide modern state-of-the-art laboratory equipment and space, including BSL-3 and BSL-4 containment facilities for working with these potentially hazardous agents.The WRCEVA provides prompt analysis of disease outbreaks as well as identification of new and emerging viruses from field collected samples  provided by agencies around the world. Since its inception, the WRCEVA has been in the forefront of virus discovery and characterization. For example, focusing just on the past 20 years, WRCEVA’s expertise and resources were instrumental in identifying the geographic source of the New York introduction of West Nile virus, and following its westward spread in the U.S. The WRCEVA assisted the Harris County (Houston) Mosquito Control district in the surveillance of West Nile virus in East Texas, testing thousands of mosquito pools and dead birds, an association that continues to this day ,18,19. AIn many of these discoveries, transmission electron microscopy (TEM) was essential to determine the taxonomic position (family) of the virus, which will be demonstrated by the following examples.Paramyxoviruses form at the cell surface using plasmalemma as their envelope. Usually they are pleomorphic or spherical in shape, varying in size from 100 nm to several hundred nanometers, but they can also be finger-like or filamentous A. FilameRhabdoviruses have characteristic bullet or finger-like morphology with cross-striations 5–7 nm in periodicity, reflecting helical packaging of the nucleocapsid. In cross-sections, they appear ring-like in structure C. The siNyamiviridae includes six genera. Nyamanini virus (NYMV), Midway virus, and Sierra Nevada virus (SNVV) were recently assigned to the genus Nyavirus [The family Nyavirus ,18,19. TOrthomyxoviridae are predominantly tick-borne and recently classified into the genera Thogotovirus and Quaranjavirus [Thogotovirus currently comprises 2 viruses: Thogoto virus (THOV) and Dhori virus (DHOV). Probable members also include Batken virus and its variants, Araguari virus (ARAV) [Arboviruses assigned to the family njavirus . The gens (ARAV) , Jos virs (ARAV) , Upolu vs (ARAV)  and the s (ARAV) .As for other orthomyxoviruses, virions form at the cell surface. THOV and DHOV exhibit significant variations in morphology: they can be pleomorphic, spherical or filamentous, 65 nm in diameter and up to 300 nm long. Other viruses that may be assigned to this genus are mostly spherical but display some polymorphism in sizes: virions of ABV can be 75–200 nm in diameter A, UPOV −Quaranjavirus currently includes two named viruses: Quaranfil virus (QRFV) and Johnston Atoll virus (JAV). Lake Chad virus (LKCV) [The genus s (LKCV) , Wellfles (LKCV)  and Tjuls (LKCV)  are alsos (LKCV) . The mors (LKCV) ,57,58. VBunyaviridae), we illustrate the morphology of new and emerging tick-borne viruses that are likely to be assigned to the genus Phlebovirus. Phleboviruses are spherical, enveloped viruses 75–95 nm in diameter  and are located inside the cisterns of granular endoplasmic reticulum within infected cells . Replica nm and aReoviridae includes two subfamilies, Spinareovirinae and Sedoreovirinae, according to morphology of virion surface which can be visualized in negatively stained preparations of purified viruses. Virions of viruses assigned to the Spinareovirinae have short flat spikes or turrets. In Fako virus , recently isolated from mosquitoes in Cameroon [Sedoreovirinae, virions are devoid of spikes but can display round surface capsomere subunits ~7 nm in diameter (Coltivirus) and orbiviruses (genus Orbivirus), virus factories are often associated with randomly distributed fibrils and/or microtubules as demonstrated by F.A. Murphy [In ultrathin sections of infected cells virions of arthropod-borne reoviruses appear as spherical particles 45–70 nm in diameter with central dense cores A. FamilyCameroon , these tdiameter B. Reovirdiameter A. In col. Murphy ,63.Arenavirus virions are spherical and pleomorphic with variable sizes, from 80 nm to 200 nm in diameter B. They aAlphavirus virions are icosahedral particles which appear spherical in ultrathin sections, 50-70 nm in diameter with dense nucleocapsid core C. AlphavNidovirales, being distantly related to coronaviruses . They appear to be very common and widespread in mosquito populations from different geographical and ecological locations [Mesoniviruses were recently classified as a family ,72 in thocations . Mature ocations .Negevirus) related to genera of mite-infecting plant viruses  in the new family Kitaviridae [Entomobirnavirus, family Birnaviridae . They appear to be very common and widespread in insect populations on different continents as isolates have been obtained from pools of mosquitoes and sandflies collected in Israel, North and South America, Africa, and Indonesia. In negatively stained preparations of Piura virus, isolated from Culex sp. mosquitoes in Peru, spherical particles with diameters of ~45 nm and ~55 nm were found. The striking feature of negevirus infection of C6/36 mosquito cells are the cytopathic effects – enormous expansions of perinuclear space – a granular endoplasmic reticulum system which becomes filled with vesicles and/or microtubules  (virions 55 nm in diameter inside ER cisterns) and an unknown flavivirus . Alphamesonivirus; virions 55 nm in diameter) and an unknown reovirus (virions 45 nm in diameter with a characteristic dark nucleocapsid core). We have also observed co-infection with Bontag Baru virus  and an unknown flavivirus, Ngewontan  and Nam Dinh virus (genus Alphamesonivirus); Eilat virus (genus Alphavirus) (Negevirus) (Alphamesonivirus), an unknown flavivirus and an unknown rhabdovirus, the latter budding into an ER cistern occupied by a flavivirus virion. It is most likely that these viruses originate from different individual mosquitoes in the pool, but the possibility of a co-infection of one mosquito with several different viruses cannot be excluded.Sometimes, after inoculation of C6/36 mosquito cells with homogenates of mosquito pools, several viruses can be observed in the culture, or even in the same cell. havirus) B,C and Ngevirus) A. Three The WRCEVA collection at the University of Texas Medical Branch in Galveston, Texas, plays an important role in virology research as a depository of natural viral biodiversity, and as a valuable resource for knowledge of viruses that are not only pathogenic for animals and humans but also are naturally occurring in arthropods. Propagation in cell cultures and subsequent examination of virus morphology by electron microscopy have been useful initial steps in the identification and characterization of novel viruses, giving indications for their further genetic characterization.


Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages  of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.Usutu virus (USUV) is a flavivirus ( Flaviviridae, genus Flavivirus) transmitted by ornithophilic mosquitoes. The virus was isolated from a number of mosquito species of the genera: Aedes, Anopheles, Culex, Culiseta, Ochlerotatus summarized in [Culex spp. [Pica pica), domestic pigeon (Columba livia domestica), hooded crow (Corvus cornix), carrion crow (Corvus corone), Eurasian nuthatch (Sita europea), great tit (Parus major)) and partially migratory or migratory , song thrush (T. philomelos), white stork (Ciconia ciconia), common redstart (Phoenicurus phoenicurus), and European robin (Erithacus rubecula)) bird species are considered the probable natural (reservoir) hosts in Europe, based on serologic evidence and direct proof of the virus [Usutu virus (USUV) is a flavivirus (family rized in ), hence lex spp. ,3,4,5. Ahe virus ,6,7, althe virus , rodentshe virus . Passerihe virus ,12,13,14he virus ,16,17 orhe virus . Predomihe virus ,20,21.Like other flaviviruses, the genomic +ssRNA of USUV encodes three structural proteins forming viral capsid (protein C), membrane (prM/M) and envelope (E). The remaining part of the genome encodes seven non-structural proteins  ,23. ProtCx. naevei in 1959 [Cx. modestus mosquitoes sampled in 2013 in South Moravia [Usutu virus was first isolated in African Swaziland from a mosquito  in 1959 . In Euro in 1959 . The fir in 1959 . Thereaf in 1959 . Current in 1959 . In the  in 1959  and in a Moravia .Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 USUV strains from carcasses of blackbirds using cell culture. This data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.There are reports of multiple USUV outbreaks mainly among blackbirds from other European countries e.g., ,31,32,33Turdus merula) were collected in three cities (and their surroundings) in the Czech Republic: Prague (2018), Brno (2017–2019) and Ceske Budejovice (2017)  were collected and analyzed (Cadavers of blackbirds (e (2017) . In Praganalyzed .The cadavers had been exposed for an unknown time to outdoor temperatures before collection. The place, time, date of collection, and conjectured cause of death were recorded. Cadavers were stored at −80 °C until necropsied.w/v) suspension in lysis RLT buffer  containing beta-mercaptoethanol. Stainless-steel beads of 5-mm (Qiagen) and Tissue Lyzer II (Qiagen) were used for tissue homogenization. After mechanical disruption (30 Hz for 30 s for liver and 2 min for muscle and brain tissue), the homogenate was digested with 20 µL of proteinase K for 30 min at 56 °C. The lysate was clarified by centrifugation and supernatant was collected. The blood samples were resuspended directly in 560 µL of AVL buffer. From each sample, 140 µL were used for RNA isolation using the Qiamp Viral RNA Mini Kit (Qiagen) according to manufacturer’s instructions, modified as follows. On column RNase-free DNase I (Qiagen), treatment was performed for 15 min at room temperature after the first washing step and an additional AW1 wash step was included after the digestion. The RNA was eluted with 60 µL of AVE buffer. Subsequently, the membrane was washed once more with the eluate to increase the yield of RNA. Spare aliquots of PCR positive tissue samples were used for virus isolation attempts in cell cultures.Samples of brain, liver, muscle, and blood were collected using sterile instruments for each tissue in the individual cadavers. Two specimens of each tissue (for RNA extraction and virus isolation) were created and stored at −80 °C or processed immediately. One specimen of each tissue  was used to prepare 30% . The reaction was performed according to manufacturer’s instructions in a total volume of 25 µL. Universal flavivirus primers  in 0.4 µCx. pipiens females sampled in South Bohemia, near Lomnice nad Luznici, in 2018; sample 3Cx_16-99 was obtained from a pool of 52 Cx. modestus females sampled in South Moravia, near Hlohovec, in 2016 and sample 1TM10Bre was obtained from a brain tissue of a dead blackbird found in South Moravia, town of Breclav in 2018  were subjected to cDNA synthesis using the 1st Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. Random hexamer primers and 4 µL of template total RNA were used for reverse transcription. Selected portions of the USUV genome were sequenced using Sanger sequencing primer walking strategy  or C6/36 medium . The cells were cultivated for 1 day in 0.5% of CO2 at 37 °C (PS) or 28 °C (C6/36), respectively. Brain tissue samples were homogenized in the culture media using stainless-steel beads for 1 min 30 Hz in cooled blocks of Tissue Lyzer II (Qiagen) to prepare a 30% (w/v) suspension. The suspension was clarified by centrifugation , supernatant was filtered using 0.2 µm syringe filters (VWR), and 100 µL of the filtrate were added to the prepared cells in the plate. The same volume of media used for homogenization was added in negative control wells. After 3 h of incubation at appropriate temperature (0.5% of CO2), the medium was removed, cells were washed by 3 mL of phosphate-buffered saline (PBS), 3 mL of fresh medium was added, and the cells were further cultivated. On days 1, 2, and 5 post infection, 2 mL of culture medium were sampled and replaced by fresh medium. The identity of the virus was confirmed by RT-PCR and sequencing. Virus titers were determined by plaque assay using PS cell culture, and a virus antigen was detected using a flavivirus-specific immunofluorescence staining, as described previously [Brain samples of the USUV RNA positive individuals were used for isolation of the replicating virus using cell cultures. Mammalian PS (porcine kidney)  (Kozuch eviously .T. merula), 20 (36%) were positive for flavivirus RNA. All positive findings were subsequently confirmed as USUV by sequencing and Blast analysis. From the total of eight individuals collected in the area of Prague, six were positive for USUV RNA. Another 14 positive blackbirds (17%) were found among randomly sampled bird cadavers in the area of Brno, whereas no positive animal was acquired in Ceske Budejovice. Of the 14 positive individuals from Brno, 9 were sampled in 2017 and 5 in 2018. All of the USUV positive blackbirds had detectable viral RNA in brain tissue. Detailed results including a comparison of different tissues are summarized in T. philomelos) were positive for flavivirus RNA.From the total of 55 blackbirds . From the prM/E protein section, we have determined an almost complete coding sequence of prM, whole coding sequence of M protein and E protein (nucleotide positions 562–2475). Furthermore, partial nucleotide coding sequences for viral NS4B peptide and complete coding regions of protein NS5 were determined . In the case of the sample 001TM10Bre acquired from a dead blackbird from South Moravia in 2018, only the complete NS4B/NS5 section was obtained.T. merula collected in Brno and sequences obtained from both the USUV positive mosquito pools were placed into lineage Europe 2  and mosquito (C6/36) cell lines. We have isolated USUV in 10 out of 12 samples (six of Europe 1 lineage and four of Europe 3) with the same results obtained in both cell culture systems. The active replication of the virus was confirmed using plaque assay . In PS cells, average virus titer in culture media reached 4.83 ± 0.79 log10 pfu/mL on day-2 post-infection (p.i.) and 2.27 ± 1.11 log10 pfu/mL on day-5 p.i. In C6/36 cells, average virus titer in culture media reached 3.11 ± 1.37 log10 pfu/mL on day 2 p.i. and 6.96 ± 0.61 log10 pfu/mL on day 5 p.i. In PS cells, the viral strains generated a cytopathic effect, whereas in mosquito C6/36 cells, no cytopathic effect was observed a,b. ImmuAll the isolated strains were deposited in the Arbovirus collection of the Biology Centre Collection of Organisms (BCCO) under following identification numbers: BCCO_50_0480 to BCCO_50_0501 [In the Czech Republic, the occurrence of USUV has been reported in isolated cases and restricted to the southeastern part (South Moravia) of the country ,30 neighInterestingly, we were able to detect USUV RNA also in randomly collected dead blackbirds in the city of Brno in the area of previous occurrence, where, however, no mass mortality of blackbirds was observed. Phylogenetic analyses showed that these viral strains belong to lineages Europe 1 and 2. The Europe 1 lineage virus was found previously in this region  and bothIn some areas, USUV probably may circulate unnoticed without causing any major mortalities in (urban) populations of birds. This hypothesis is supported by the detection of the virus in a pool of mosquitoes collected in South Bohemia, the southwestern part of the country close to the city of Ceske Budejovice, where no USUV positive birds were found and no mass mortality among birds was noticed.For the first time, an USUV strain of Africa 3 lineage was found in the Czech Republic in a dead blackbird from South Moravia region (found in the town of Breclav). USUV strains of Africa 3 and Africa 3-like lineages were repeatedly detected in Europe  includinT. philomelos) were positive in our study, despite the virus which was detected in this species previously [Carcasses of dead birds represent an important source of material for surveillance of USUV and other ornithophilic viruses ,51. As reviously ,32,53.Cx. pipiens and in a pool of Cx. modestus mosquitoes. Cx. pipiens is considered a principal vector of USUV in Europe [Cx. modestus [Cx. modestus (together with Cx. pipiens) in the maintenance of USUV in a sylvatic cycle. Cx modestus is a human biting species and is considered a potential bridge vector of the related West Nile virus (WNV) to humans [USUV was also detected in a pool of n Europe , multipln Europe ,3, and tn Europe ,55,56. Wmodestus . The auto humans . Nevertho humans .In conclusion, USUV presence in the Czech Republic is not restricted to the southeastern part of the country neighboring with Austria. Two genetic lineages (Europe 1 and Europe 2) probably co-circulate in this area of endemic occurrence without causing any major outbreaks in wild life. Lineage Europe 3 was probably newly introduced to the area of Prague and resulted in a local mortality of blackbirds.


Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the “perfect storm” that would ultimately lead to a spillover. One of these factors is the increased shedding of virus by bats. Several studies have indicated that bats have unique defense mechanisms that allow them to be persistently or latently infected with viruses. Factors leading to an increase in the viral load of persistently infected bats would facilitate shedding of virus. This article reviews the unique nature of bat immune defenses that regulate virus replication and the various molecular mechanisms that play a role in altering the balanced bat–virus relationship. Over the past few years, the interest in bat research has increased because of the range of viruses that they harbor and the potential of spillover to humans and other vertebrates . In somePathogen shedding by bats is a complex phenomenon regulated by host antiviral defenses and viral factors. A host’s defenses attempt to control virus infection but viruses have evolved mechanisms to counteract them . When thTherefore, it becomes important to study what factors might be responsible for decreasing the host defense or factors that allow the virus to circumvent host immune response. But before exploring factors affecting virus–host interactions, it is imperative to understand the viruses present in bats and the bat immune defenses. In 2013–2014, bat immunology was at a nascent stage and Schountz and Baker et al.  had summPteropus alecto), the interferon gene locus is contracted, whereas in the Egyptian fruit bat (Rousettus aegyptiacus), this locus is expanded [One must be cautious when interpreting the literature on bat defenses. The order Chiroptera contains more than 1200 species . Due to expanded ,19. OtheIt has been observed that viruses which severely affect other mammals, including humans, are apparently nonpathogenic for bats . This adMyotis davidii) [The immune response has two primary components, innate and adaptive. The host initially responds to infection by activating innate mechanisms. Genes such as those for sensing and repairing DNA damage and the inflammatory process are under positive selection in black flying foxes and David’s myotis (davidii) . For insdavidii) ,23. It idavidii) . In contdavidii) . Novel mArtibeus jamaicensis) [Cruz-Rivera et al.  showed ticensis) . Inactivicensis) .These studies suggest that in bats of several species, higher level of interferon and ISGs are always present in their cells, which makes them better prepared to control viruses. Overall, bats seem to possess either an “always ON” interferon strategy plus or a better antiviral ISG defense strategy.Eptesicus fuscus), stimulated with poly I:C, a surrogate for double-stranded viral RNA, cRel actively suppresses TNFα expression [While bats are well prepared to control viral infections, they also have mechanisms to avoid over-induction of inflammatory genes. Such excessive inflammation is detrimental in vertebrates of other species, such as humans, and is linked to pathology . So, it pression . Genome pression . This suSeveral species of bats control inflammation by having a mutation at a highly conserved serine residue in one of the key adaptor molecules for sensing damaged DNA, i.e., stimulator of interferon genes (STING), which reduces its functionality. STING senses damaged DNA or dsDNA from viruses and induces an interferon response. The replacement of serine at position 358 by other amino acids makes bat STING less effective in activating interferons . In addiThe unique features of bats, whereby they tolerate viral infections without excessive inflammation while suppressing viral replication, leads to the obvious question: What is so special about bats? The answer to this has been linked to the evolution of the ability to fly. The increased rate of metabolism accompanying flight would lead to higher levels of oxygen-free radicals ,33. ThisOver the past few years, numerous viruses have been detected in bats and these viruses seldom cause any overt disease (with the exception of Tacaribe virus and rabies virus) ,16,37. DPteropus vampyrus), showed the possibility of recrudescence of Nipah virus (NiV). Regular sampling was done for over a period of one year to study the presence of virus and level of antibody against it. The authors discovered that one bat had a waning antibody titer with subsequent detection of virus in the urine. Following virus shedding, the antibody levels increased in this animal. As virus was undetectable in earlier samples of the same bat, the authors suggest that the virus may have persisted, probably in certain organs or cells, rendering it undetectable in blood, throat swab or urine. Within two weeks after NiV was isolated from the bat, two other male bats seroconverted and demonstrated an increase in antibody titers, suggesting that recrudescence led to horizontal transmission.A study done by Sohayati et al. , done onR. aegyptiacus) showed that bats naturally infected by other experimentally inoculated bats, seem to have a prolonged incubation period [Other studies on Marburg virus transmission among Egyptian rosette bats , and a Myotis lucifugus) [A bat coronavirus was also shown to persistently infect North American little brown bats (cifugus) . Little Eidolon helvum). While repeated introduction of virus and birthing of pups might drive viral dynamics in a large panmictic population of bats, prolonged infectious periods or latent infection of bats are required to explain henipavirus persistence in small populations . They found that if repeated introduction of virus into small populations was the only mechanism, then acute infections would have to be ~40 days in duration. This estimate is considerably longer than the current estimates of the detectable infectious period for henipaviruses in fruit bats, which is approximately 7 days [To further bolster the claim of viral persistence in wild bats, a population level study was performed to understand the circulation of zoonotic viruses in bat populations and the involved immune mechanisms  using mathematical modelling . The stuy 7 days . TherefoSpillover events are complex and usually require successful alignment of several contributing factors . One suc+ T cells which secrete interferon-γ (IFN-γ) and suppress viral transcription factors via noncytolytic granzyme mediated degradation [+ T cells capable of producing IFN-γ. This contributes to reactivation of the virus [Several factors may alter long-term, low-level viral persistence in bats. Suppression of the immune response holding active virus replication in check would allow the virus to replicate to higher levels. This could happen in stressful conditions that affects the immune system. In other animals, a variety of stressors lead to reactivation of latent herpesviruses as reviewed by Grinde ,49. In nradation . During he virus . Studieshe virus ,53. Strehe virus .Arousal from hibernation is a stressful event for bats . Many biIn pteropid bats, immunological stress was suggested as a contributing factor in henipavirus shedding . As descPseudogymnoascus destructans [Secondary infections in humans are immunologically stressful . Little tructans ,60,61. Atructans . The intThese studies indicate that waning antibody levels and suppression of innate immune response due to stress might be some of the factors leading to an increase in viral levels in persistently infected bats .The unique features of bat immune responses that promote viral persistence may exert evolutionary pressures on the virus as well. Bats have superseded rodents in harboring greater number of viruses and also having greater proportion of zoonotic viruses . It is tAlthough there is some evidence for the increase in virus replication and shedding in bats under stress, a direct link of this to spillover events has yet to be discovered. Future controlled experiments aimed at studying transmission dynamics in the presence and absence of stress in bats would lead to a more definitive answer. It is also important to look into various factors that might stress bats such as habitat destruction (deforestation), pregnancy, change in seasons, and climate change. Additionally, the molecular mechanisms leading to the waning of antibodies and other aspects of adaptive immune response in bats are not known. A holistic picture of bat immune systems and the factors leading to an increase in viral replication might help us further understand viral spillovers.


We previously developed CCR5-tropic neutralization-resistant simian/human immunodeficiency virus (SHIV) strains and a rhesus macaque model of infection with these SHIVs. We induced the production of neutralizing antibodies (nAbs) against HIV-1 by infecting rhesus macaques with different neutralization-resistant SHIV strains. First, SHIV-MK1 (MK1)  with CCR5 tropism was generated from SHIV-KS661 using CXCR4 as the main co-receptor. nAbs against parental-lineage and heterologous tier 2 viruses were induced by tier 1B virus (MK1) infection of the rhesus macaque MM482. We analyzed viral resistance to neutralization over time in MM482 and observed that the infecting virus mutated from tier 1B to tier 2 at 36 weeks postinfection (wpi). In addition, an analysis of mutations showed that N169D, K187E, S190N, S239, T459N (T459D at 91 wpi), and V842A mutations were present after 36 wpi. This led to the appearance of neutralization-resistant viral clones. In addition, MK1 was passaged in three rhesus macaques to generate neutralization-resistant SHIV-MK38 (MK38) (tier 2). We evaluated nAb production by rhesus macaques infected with SHIV-MK38 #818 (#818) (tier 2), a molecular clone of MK38. Neutralization of the parental lineage was induced earlier than in macaques infected with tier 1B virus, and neutralization activity against heterologous tier 2 virus was beginning to develop. Therefore, CCR5-tropic neutralization-resistant SHIV-infected rhesus macaques may be useful models of anti-HIV-1 nAb production and will facilitate the development of a vaccine that elicits nAbs against HIV-1.The online version of this article (10.1007/s00705-019-04173-5) contains supplementary material, which is available to authorized users. Antiretroviral agents are used against human immunodeficiency virus type 1 (HIV-1), but eliminating latent HIV-1 is difficult . Therefoenv gene of HIV-1, can be used to evaluate nAbs against the Env protein of HIV-1.The rhesus macaque model of simian immunodeficiency virus (SIV) infection is important as an animal model of AIDS for pathogenicity studies and vaccine development. However, the envelope protein (Env) of SIV has a low level of amino acid sequence similarity to that of HIV-1 , and nAbet al. [et al. [Controlling HIV and SIV is difficult, as they use CCR5 as a co-receptor; however, SHIV-89.6P (CXCR4) is easy to control . Seaman et al.  reportedet al. . SHIV-SFet al. , 41–46. et al. –50 and tet al. . Ishida  [et al.  producedIn this study, we evaluated nAb production by rhesus macaques infected with CCR5-tropic tier 1 and tier 2 SHIV. nAbs against tier 2 virus were induced by tier 1B virus infection, and production of nAbs against tier 2 virus began earlier in Tier 2 virus infection. Our findings provide important insights that might be applicable to HIV-1 vaccine development.2.HEK293T (293T) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum . TZM-bl cells were cultured in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM sodium pyruvate  and 4 mM L-glutamine . Cells were harvested and passaged using trypsin/ethylenediaminetetraacetic acid solution  and were maintained at 37 °C in a humidified atmosphere containing 5% COin vivo passages from macaque M482 to macaque MM498 (SHIV-MK1-first passage), and subsequently to macaque MM504 (SHIV-MK1-second passage). This enhanced viral replication and the re-isolated virus was designated SHIV-MK38 (MK38). Next, we inoculated MK38 intravenously into rhesus macaques  [SHIV-MK1, SHIV-MK1-first passage, SHIV-MK1-second passage, and SHIV-MK38 were described previously , as was d MM502) . The mold MM502) . We mimid MM502) . R5 virud MM502) , 56. Indenv gene of MK1, #818, murine leukemia virus (MLV), or clade B panel viruses (NIH AIDS reagent program) were prepared by co-transfecting 293T cells with pSGΔenv and pcDNA3.1 vectors expressing the respective env genes. We obtained pSGΔenv, pcDNA3.1 vectors expressing clade B env, and vectors expressing MLV env from the NIH AIDS reagent program. To construct the pcDNA3.1 vector expressing the rev and env genes of MK1 and #818, approximately 3.0 kb of the region including the rev and env genes of pMK1 [env-deleted region from pcDNA3.1-SHIVMNA [env was generated by InFusion cloning using the pcDNA3.1 vector and SHIV-MNA env PCR product) was amplified by PCR using the primers SHenv7F (GGGACAGTATATGAATACTCC) and IFrevR (ATAGGAGATGCCTAAGGC). PCR was performed as follows: 1 cycle of denaturation , 30 cycles of amplification , and a final extension . The buffer and polymerase were as above. The PCR products were purified using a NucleoSpin Gel and PCR Clean-up Kit , and env-depleted pcDNA3.1 was reacted with the inserted env DNA. Cloning was conducted using an In-Fusion HD Cloning Kit (TaKaRa), and the resulting plasmid DNA was introduced into Stbl3 cells by electroporation.Pseudotype viruses harboring the  of pMK1  and pMK3 of pMK1  were amp-SHIVMNA   according to the manufacturer’s protocol. cDNA, including the env gene, was synthesized from the extracted RNA by reverse transcription using random hexamers  and SuperScript IV Reverse Transcriptase (Invitrogen). To construct the pcDNA3.1 vector expressing the rev and env genes of viruses obtained from MM482, at weekly intervals after infection, approximately 3.3 kb of the region including the rev and env genes in the cDNA template was amplified by PCR using the primers SHenv0F (AGAGCAAGAAATGGAGCCAG) and SHenv8.5R (CCATAGCCAGCCAAATGTCT). PCR was performed as follows: 35 cycles of amplification  using KOD One PCR Master Mix (Toyobo), 15 µM primers, and template. Next, approximately 2.9 kb of the region including the rev and env genes in the PCR product was amplified by nested PCR using the primers InsertF3 (TTCACCGGCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGA) and InsertR3 (TTGACCACTTGCCCCCCATTTGTCCCTCACAAGAGAGTGAGCT). PCR was performed as above. The PCR products were purified using a NucleoSpin Gel and PCR Clean-up Kit (TaKaRa) and sequenced directly . Approximately 5.5 kb of the env-deleted region from pcDNA3.1-SC422661  were amplified by PCR using the primers VectorF3 (AATGGGGGGCAAGTGGTCAA) and VectorR3 (AGGAGATGCCTAAGCCGGTGAA). PCR was performed as follows: 35 cycles of amplification; 98 °C for 10 s, 58 °C for 5 s, and 68 °C for 27 s. The buffer and polymerase were as above. The PCR products were purified, and env-depleted pcDNA3.1 was reacted with the inserted env DNA. Cloning was conducted using an NEBuilder HiFi DNA Assembly Master Mix , and the resulting plasmid DNA was introduced into Stbl3 cells by electroporation.Pseudotype viruses harboring the 50) was determined with the infectivity of wells lacking plasma defined as 100%. A high ID50 value thus indicates potent inhibition. Plasma from infected macaques was inactivated at 56 °C for 60 min and centrifuged at 11,000 rpm for 10 min. The pooled plasma of HIV-1-infected individuals and infected macaques was diluted in fourfold steps from 1:50 to 1:204,800 and pre-incubated with virus (100 TCID50) at 37 °C for60 min. Next, 5,000 TZM-bl cells were cultured with the pre-incubated mixture in the presence of 5 mg of DEAE dextran/mL at 37 °C for 48 h. To measure luciferase activity, 50 of µL cell lysate solution  was added to each well and the plate was agitated for 15 min. An aliquot of 30 µL of lysate was transferred to a Nunc F96 MicroWell white plate , and the luminescent substrate (50 µL) was added to each well. Luciferase activity was calculated with Mikrowin and a TriStar LB 941 reader . ID50 values were calculated as described previously [Neutralization assays were performed using various pseudoviruses with pooled plasma from HIV-1-infected patients  as a positive control. Luciferase activity was measured in TZM-bl cells . Plasma eviously .50 values were calculated as previously described [As an anti-HIV-1-neutralizing monoclonal antibody, we used KD-247 . KD-247 was diluted fourfold from 20 to 0.005 µg/mL, and ICescribed .6–108 copies/mL and was maintained at 103–104 copies/mL in MM482. In MM498, MM504, MM481, MM501, and MM502, the plasma viral RNA level peaked at 107–108 copies/mL and was maintained at 104–107 copies/mL in all of these macaques. In MM596, MM597, and MM599, the plasma viral RNA level peaked at 107–108 copies/mL and was maintained at 105–106 copies/mL only in MM597. Seven of the ten rhesus macaques developed persistent infections. Many HIV-1-infected patients have a persistent infection with neutralization-resistant virus [In MM482 and MM483, the plasma viral RNA level peaked at 10nt virus . To deveenv as the negative control. Potent neutralization of MK1, equal to or higher than that of HPP , was detected in MM482, MM504, MM501, MM502, and MM597 . Additionally, neutralization activity against #818 higher than that of HPP  was detected in MM504, MM501 and MM597   from HIV-1-infected patients as the positive control and a pseudovirus containing mouse leukemia virus (MLV) P ID50, 81, was deP ID50, 81, was de50, 345 and 192, respectively)  , and antibodies were induced against SF162 at 6 wpi  , and MK 1 (tier 1 B virus). The GPGR epitope of KD 247 was preserved in all viruses from 5 to 115 wpi in the plasma of MM482. Clones 3, 4, 5, 6, 8, 9, and 10 were prepared from 12 wpi plasma. Clones 6 and 10 contained minor amino acid mutations. All of the clones showed greater resistance to neutralization than the tier 1 B MK 1 virus, but less resistance than that of the tier 2 #818 virus. At 12 wpi, all clones  remained tier 1B  , 52. Theenv sequence diversity increases. Subsequently, the host humoral immune response results in production of nAbs with increased affinity. After a number of years, some patients produce antibodies that target one or more shared epitopes, resulting in cross-reactivity with heterologous strains. This leads to induction of broadly neutralizing antibodies with activity against diverse tier 2 viruses [In MM597, nAbs against parental-lineage tier 2 viruses were rapidly induced, and nAbs against heterologous tier 2 viruses were beginning to be induced Tables . In HIV- viruses . TherefoKS661 was susceptible to neutralization (tier 1B). Induction of Abs in macaques infected with KS661 inhibits viral replication; however, MK38 became resistant to neutralization tier 2) . MK38 an . MK38 aIn MM482, the N169D, K187E, S190N, S239, T459N (T459D at 91 wpi), and V842A mutations were detected at 36 wpi. Because the tier 2 #818 virus has three mutations in the V2 region  , the above-mentioned six mutations likely contribute to neutralization resistance.50 values were low -infected rhesus macaques will enable analysis of the neutralization resistance of viruses that induce tier 2 nAbs and the antigen needed to induce broadly neutralizing antibodies. Finally, these animal models will facilitate the development of HIV-1 vaccines.Supplementary material 1 (PDF 58 kb)Below is the link to the electronic supplementary material.


Mops condylurus) in Kenya. Complete genome analysis revealed 98% nucleotide sequence similarity to the prototype virus from Sierra Leone. No Ebola virus–specific RNA or antibodies were detected from febrile humans in the area who reported contact with bats.Bombali virus (genus Ebolavirus) was identified in organs and excreta of an Angolan free-tailed bat ( Filoviridae is divided into 5 genera: Cuevavirus, Marburgvirus, Ebolavirus, Striavirus, and Thamnovirus . Six distinct Ebola viruses have been described; 4 are known to cause human disease  captured near the Taita Hills in southeastern Kenya, the easternmost distributional range of this bat species  and was reported in August 2018 in mouth and fecal swabs collected from free-tailed insectivorous bat species (family Molossidae)  species . We alsoM. condylurus and is not an artifact of its insect diet, which could not be discounted from the previous analysis on the basis of mouth and fecal swabs (C. pumilus bats (n = 13) and other bat species  by reverse transcription PCR and next-generation sequencing. This bat was captured along with 15 others in mist nets in savannah habitat near a small river in May 2018; only this bat was BOMV positive; . Viral RNA was present in lung, spleen, liver, heart, intestine, mouth swab, and fecal samples but absent from the brain, kidney, urine, and a few fleas found on the bat; viral loads were especially high in the lung . These t species  Table 2  species  Figure,  species .M. condylurus bats; it is possible that BOMV–positive findings from other bat species result from local spillover or contamination. Moreover, phylogenetic analysis of the full BOMV genome from the bat lung revealed 98% nucleotide sequence similarity with the prototype reported in Sierra Leone (GenBank accession no. MK340750)  . ConsideM. condylurus bats commonly roost in human structures, such as house roofs (,Because e roofs ,. We scree roofs ,. Many saM. condylurus bat as a competent host. Like Goldstein et al. (,Our results markedly expand the distributional range of this new Ebola virus to eastern Africa and confirm the Mops condylurus bat, Kenya.Additional information about Bombali Ebola virus in