Surveillance and Genetic Analysis of Jamestown Canyon Virus in New York State: 2001-2022.

Jamestown Canyon virus (JCV) (Peribunyavirdae; Orthobunyavirus) is a mosquito-borne pathogen endemic to North America. The genome is composed of three segmented negative-sense RNA fragments designated as small, medium, and large. Jamestown Canyon virus is an emerging threat to public health, and infection in humans can cause severe neurological diseases, including encephalitis and meningitis. We report JCV mosquito surveillance data from 2001 to 2022 in New York state. Jamestown Canyon virus was detected in 12 mosquito species, with the greatest prevalence in Aedes canadensis and Anopheles punctipennis. Detection fluctuated annually, with the highest levels recorded in 2020. Overall, JCV infection rates were significantly greater from 2012 to 2022 compared with 2001 to 2011. Full-genome sequencing and phylogenetic analysis were also performed with representative JCV isolates collected from 2003 to 2022. These data demonstrated the circulation of numerous genetic variants, broad geographic separation, and the first identification of lineage B JCV in New York state in 2022.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Identification and characterization of novel lineage 1 Powassan virus strains in New York State.

Powassan virus (POWV, family Flaviviridae) is a reemerging tick-borne virus endemic in North America and Russia. In 1997, a POWV-like agent was isolated from Ixodes scapularis in New England and determined to be genetically distinct from the original POWV isolate. This revealed the existence of two lineages: lineage 1, prototype Powassan virus (POWV-1) and lineage 2, deer tick virus (DTV). POWV-1 is thought to be primarily maintained in a cycle between I. cookei and woodchucks and I. marxi and squirrels, while DTV is primarily maintained in a cycle between I. scapularis and small mammal hosts. Recent tick, mammalian, and human isolates from New York State (NYS) have been identified as DTV, but for the first time in 45 years, we detected four POWV-1 isolates, including the first reported isolation of POWV-1 from I. scapularis. We aimed to investigate genotypic and phenotypic characteristics of recent NYS isolates through sequence analysis and evaluation of replication kinetics in vitro and in vivo. Our sequencing revealed genetic divergence between NYS POWV-1 isolates, with two distinct foci. We found that POWV-1 isolates displayed variable replication kinetics in nymphal ticks but not in cell culture. POWV-1 isolated from I. scapularis displayed increased fitness in experimentally infected I. scapularis as compared to historic and recent POWV-1 isolates from I. cookei. These data suggest the emergence of divergent POWV-1 strains in alternate tick hosts and maintenance of genetically and phenotypically discrete POWV-1 foci.

Local connectivity and synaptic dynamics in mouse and human neocortex.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Aedes Albopictus and Cache Valley virus: a new threat for virus transmission in New York State.

We report surveillance results of Cache Valley virus (CVV; Peribunyaviridae, Orthobunyavirus) from 2017 to 2020 in New York State (NYS). Infection rates were calculated using the maximum likelihood estimation (MLE) method by year, region, and mosquito species. The highest infection rates were identified among Anopheles spp. mosquitoes and we detected the virus in Aedes albopictus for the first time in NYS. Based on our previous Anopheles quadrimaculatus vector competence results for nine CVV strains, we selected among them three stains for further characterization. These include two CVV reassortants (PA and 15041084) and one CVV lineage 2 strain (Hu-2011). We analyzed full genomes, compared in vitro growth kinetics and assessed vector competence of Aedes albopictus. Sequence analysis of the two reassortant strains (PA and 15041084) revealed 0.3%, 0.4%, and 0.3% divergence; and 1, 10, and 6 amino acid differences for the S, M, and L segments, respectively. We additionally found that the PA strain was attenuated in vertebrate (Vero) and mosquito (C6/36) cell culture. Furthemore, Ae. albopictus mosquitoes are competent vectors for CVV Hu-2011 (16.7-62.1% transmission rates) and CVV 15041084 (27.3-48.0% transmission rates), but not for the human reassortant (PA) isolate, which did not disseminate from the mosquito midgut. Together, our results demonstrate significant phenotypic variability among strains and highlight the capacity for Ae. albopictus to act as a vector of CVV.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth's average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector-virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

Reconciling functional differences in populations of neurons recorded with two-photon imaging and electrophysiology.

Extracellular electrophysiology and two-photon calcium imaging are widely used methods for measuring physiological activity with single-cell resolution across large populations of cortical neurons. While each of these two modalities has distinct advantages and disadvantages, neither provides complete, unbiased information about the underlying neural population. Here, we compare evoked responses in visual cortex recorded in awake mice under highly standardized conditions using either imaging of genetically expressed GCaMP6f or electrophysiology with silicon probes. Across all stimulus conditions tested, we observe a larger fraction of responsive neurons in electrophysiology and higher stimulus selectivity in calcium imaging, which was partially reconciled by applying a spikes-to-calcium forward model to the electrophysiology data. However, the forward model could only reconcile differences in responsiveness when restricted to neurons with low contamination and an event rate above a minimum threshold. This work established how the biases of these two modalities impact functional metrics that are fundamental for characterizing sensory-evoked responses.

Distinct Transcriptomic Cell Types and Neural Circuits of the Subiculum and Prosubiculum along the Dorsal-Ventral Axis.

Subicular regions play important roles in spatial processing and many cognitive functions, and these are mainly attributed to the subiculum (Sub) rather than the prosubiculum (PS). Using single-cell RNA sequencing, we identify 27 transcriptomic cell types residing in sub-domains of the Sub and PS. Based on in situ expression of reliable transcriptomic markers, the precise boundaries of the Sub and PS are consistently defined along the dorsoventral axis. Using these borders to evaluate Cre-line specificity and tracer injections, we find bona fide Sub projections topographically to structures important for spatial processing and navigation. In contrast, the PS sends its outputs to widespread brain regions crucial for motivation, emotion, reward, stress, anxiety, and fear. The Sub and PS, respectively, dominate dorsal and ventral subicular regions and receive different afferents. These results reveal two molecularly and anatomically distinct circuits centered in the Sub and PS, respectively, providing a consistent explanation for historical data and a clearer foundation for future studies.

West Nile Virus fidelity modulates the capacity for host cycling and adaptation.

The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

Adaptation of Rabensburg virus (RBGV) to vertebrate hosts by experimental evolution.

Rabensburg virus (RBGV; Flaviviridae, Flavivirus) has been classified as both a novel flavivirus and a unique lineage of West Nile virus (WNV). RBGV and WNV share approximately 76% sequence homology, yet RBGV does not replicate to high viral titers within vertebrate cell lines at physiological temperatures and has not been naturally isolated from a vertebrate host. These unique genetic and biological characteristics make RBGV a viable tool to identify the genetic determinants of flavivirus infectivity and fitness in vertebrate hosts. Using experimental evolution, we characterized mutated variants of RBGV that have altered capacity for infection and replication in various cell lines. Shared genetic differences within these variants were identified throughout the genome, with a large majority found in the NS3 and NS5 genes. Our results support a role for the replication complex in host utilization and suggest that epistatic interactions likely contribute to host-specific fitness and emergence.

An anatomic transcriptional atlas of human glioblastoma.

Glioblastoma is an aggressive brain tumor that carries a poor prognosis. The tumor's molecular and cellular landscapes are complex, and their relationships to histologic features routinely used for diagnosis are unclear. We present the Ivy Glioblastoma Atlas, an anatomically based transcriptional atlas of human glioblastoma that aligns individual histologic features with genomic alterations and gene expression patterns, thus assigning molecular information to the most important morphologic hallmarks of the tumor. The atlas and its clinical and genomic database are freely accessible online data resources that will serve as a valuable platform for future investigations of glioblastoma pathogenesis, diagnosis, and treatment.

High levels of local inter- and intra-host genetic variation of West Nile virus and evidence of fine-scale evolutionary pressures.

West Nile virus (WNV; Flaviviridae, Flavivirus) has been endemic in New York State (NYS) since its 1999 introduction, yet prevalence in Culex mosquitoes varies substantially over small spatial and temporal scales. It is unclear if viral genetics plays a role in this variability, as genetic and phenotypic characterization on local scales has generally been lacking. In addition, intrahost diversity of circulating strains have not been fully characterized despite the documented role of minority variants in viral fitness and virulence. In an effort to characterize WNV variability within epidemiologically relevant scales, we performed phylogenetic analyses on NYS isolates from 1999 to 2012. In addition, we performed full-genome, deep-sequencing and genetic analyses on 15 WNV strains isolated in 2012 from Cx. pipiens in an endemic focus of Suffolk County, NY. Our results indicate continued evolution and seasonal maintenance in NYS, yet also widespread mixing and high levels of genetic diversity within geographic foci and individual seasons. Well supported local clusters with shared amino acid differences were identified and suggest local evolutionary pressures and the potential for phenotypic variability. Intrahost diversity of focal isolates was also high, with polymorphism at levels >1.0% identified in approximately 10% of the WNV genome. Although most minority mutations were unique, mutational hotspots shared among local isolates were identified, particularly in C, NS1 and NS2A genes. The most polymorphic region, positions 3198-3388 of the NS1 gene, was comprised predominately of non-synonymous mutations, suggesting a selective advantage for amino acid diversity in this region.

Consequences of in vitro host shift for St. Louis encephalitis virus.

Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. As arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are still not characterized fully for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrated that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants implicates the NS3 gene as an important host range determinant for SLEV.

Characterization of a small plaque variant of West Nile virus isolated in New York in 2000.

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.

Role of the mutant spectrum in adaptation and replication of West Nile virus.

West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.