Divergent Rabies Virus Variant of Probable Bat Origin in 2 Gray Foxes, New Mexico, USA.

In the Western Hemisphere, bat-associated rabies viruses (RABVs) have established independent transmission cycles in multiple mammal hosts, forming genetically distinct lineages. In New Mexico, USA, skunks, bats, and gray foxes are rabies reservoir hosts and represent a public health risk because of encounters with humans. During 2015 and 2019, two previously undescribed RABVs were detected in 2 gray foxes (Urocyon cinereoargenteus) in Lincoln County, New Mexico. Phylogenetic analysis of the nucleoprotein gene indicated that the isolates are a novel RABV variant. These 2 cases probably represent repeated spillover events from an unknown bat reservoir to gray foxes. Molecular analysis of rabies cases across New Mexico identified that other cross-species transmission events were the result of viral variants previously known to be enzootic to New Mexico. Despite a robust rabies public health surveillance system in the United States, advances in testing and surveillance techniques continue to identify previously unrecognized zoonotic pathogens.

Diapause influenced oviposition behavior and physical egg hatch cues of Aedes atropalpus (Diptera: Culicidae): traits that may influence successful colonization of riverine rock pools.

Mosquitoes have developed specialized oviposition strategies that allow them to develop in a wide variety of aquatic habitats. Environmentally cued hatching traits may also play an important role in the successful colonization of some larval habitats, but this subject has remained largely unexplored in Culicidae. Aedes atropalpus (Coquillett) is an autogenous rock pool specialist that may maintain unique adaptations for oviposition and egg hatching. We investigated the egg-laying strategies of Ae. atropalpus exposed to standard (non-diapausing) rearing conditions and diapause-inducing conditions and tested the impact of physical agitation on egg hatch rates by exposing floating and submerged eggs to physical agitation treatments. The results of the oviposition experiment indicate that Ae. atropalpus females primarily lay non-diapausing eggs directly onto the water surface and lay diapausing eggs directly on solid surfaces. The egg-hatching experiment demonstrated that physical agitation significantly increases Ae. atropalpus hatch rates. Floating and submerged eggs responded similarly to the agitation treatment. These data suggest that oviposition behaviors based on both egg diapause status and environmentally-cued hatching strategies may be important adaptations for Ae. atropalpus in riverine rock pools.

Aquatic thermal conditions predict the presence of native and invasive rock pool Aedes (Diptera: Culicidae) in the southern Appalachians, U.S.A.

The native rock pool mosquito, Aedes atropalpus (Coquillett), and the invasive Aedes japonicus (Theobald) have been found in many types of artificial and natural containers throughout North America. Little is known about the ecology of these two species in habitats where they co-occur, although multiple investigators have reported the decline of the native species concurrent with the introduction and spread of the invasive species. Here we report the results of riverine rock pool collections (n=503) in the southern Appalachian Mountains between 2009-2015. Surface water temperatures strongly predicted the presence of each species across a broad range of observed temperatures (11-39.8° C). For every unit of increase in temperature (°C) the odds of collecting Ae. atropalpus larvae increased by 0.34 while the odds of collecting Ae. japonicus larvae decreased by 0.28. No Ae. japonicus larvae or pupae were collected at temperatures greater than 36° C; however, immature Ae. atropalpus were found in rock pools with temperatures up to 39.8° C. In contrast, Ae. japonicus were highly abundant in cooler rock pools (<17° C) where Ae. atropalpus were infrequent or absent. Our findings suggest that in spite of the successful invasion by Ae. japonicus, Ae. atropalpus remains well established in the southern Appalachian Mountains. Given the strong correlation of temperature with the presence of the two species and the contrasting absence of each species at observed temperature extremes, the role of thermal conditions should be carefully explored in the context of other ecological factors likely influencing the range and abundance of these mosquitoes.

A paratransgenic strategy to block transmission of Xylella fastidiosa from the glassy-winged sharpshooter Homalodisca vitripennis.

BACKGROUND: Arthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control. RESULTS: Here, we report a paratransgenic strategy that targets transmission of Xylella fastidiosa, a leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter (GWSS), Homalodisca vitripennis. Earlier, we identified Pantoea agglomerans, a bacterial symbiont of the GWSS as the paratransgenic control agent. We genetically engineered P. agglomerans to express two antimicrobial peptides (AMP)-melittin and scorpine-like molecule (SLM). Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti-Xylella activity at concentrations that did not kill P. agglomerans. Using these AMP-expressing strains of P. agglomerans, we demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels. CONCLUSION: This is the first report of halting pathogen transmission from paratransgenically modified insects. It is also the first demonstration of paratransgenic control in an agriculturally important insect vector.

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order.

Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification.

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.

Highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data.

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.

West Nile virus population genetics and evolution.

West Nile virus (WNV) (Flaviviridae: Flavivirus) is transmitted from mosquitoes to birds, but can cause fatal encephalitis in infected humans. Since its introduction into North America in New York in 1999, it has spread throughout the western hemisphere. Multiple outbreaks have also occurred in Europe over the last 20 years. This review highlights recent efforts to understand how host pressures impact viral population genetics, genotypic and phenotypic changes which have occurred in the WNV genome as it adapts to this novel environment, and molecular epidemiology of WNV worldwide. Future research directions are also discussed.

West Nile virus genetic diversity is maintained during transmission by Culex pipiens quinquefasciatus mosquitoes.

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.

Molecular evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999-2008.

West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V→A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.

Environmental and biological factors influencing Culex pipiens quinquefasciatus (Diptera: Culicidae) vector competence for West Nile Virus.

Interactions between environmental and biological factors affect the vector competence of Culex pipiens quinquefasciatus for West Nile virus. Three age cohorts from two Cx. p. quinquefasciatus colonies were fed blood containing a low- or high-virus dose, and each group was held at two different extrinsic incubation temperatures (EIT) for 13 days. The colonies differed in the way that they responded to the effects of the environment on vector competence. The effects of mosquito age on aspects of vector competence were dependent on the EIT and dose, and they changed depending on the colony. Complex interactions must be considered in laboratory studies of vector competence, because the extent of the genetic and environmental variation controlling vector competence in nature is largely unknown. Differences in the environmental (EIT and dose) and biological (mosquito age and colony) effects from previous studies of Cx. p. quinquefasciatus vector competence for St. Louis encephalitis virus are discussed.

Molecular epidemiology of Powassan virus in North America.

Powassan virus (POW) is a tick-borne flavivirus distributed in Canada, the northern USA and the Primorsky region of Russia. POW is the only tick-borne flavivirus endemic to the western hemisphere, where it is transmitted mainly between Ixodes cookei and groundhogs (Marmota monax). Deer tick virus (DTV), a genotype of POW that has been frequently isolated from deer ticks (Ixodes scapularis), appears to be maintained in an enzootic cycle between these ticks and white-footed mice (Peromyscus leucopus). DTV has been isolated from ticks in several regions of North America, including the upper Midwest and the eastern seaboard. The incidence of human disease due to POW is apparently increasing. Previous analysis of tick-borne flaviviruses endemic to North America have been limited to relatively short genome fragments. We therefore assessed the evolutionary dynamics of POW using newly generated complete and partial genome sequences. Maximum-likelihood and Bayesian phylogenetic inferences showed two well-supported, reciprocally monophyletic lineages corresponding to POW and DTV. Bayesian skyline plots based on year-of-sampling data indicated no significant population size change for either virus lineage. Statistical model-based selection analyses showed evidence of purifying selection in both lineages. Positive selection was detected in NS-5 sequences for both lineages and envelope sequences for POW. Our findings confirm that POW and DTV sequences are relatively stable over time, which suggests strong evolutionary constraint, and support field observations that suggest that tick-borne flavivirus populations are extremely stable in enzootic foci.

Population variation of West Nile virus confers a host-specific fitness benefit in mosquitoes.

West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.

Larval environmental temperature and the susceptibility of Aedes albopictus Skuse (Diptera: Culicidae) to Chikungunya virus.

A key feature in the recent widespread epidemic of the mosquito-borne alphavirus chikungunya virus (CHIKV) was the important role of Aedes albopictus, formerly regarded as a secondary vector, compared to the presumed primary vector Aedes aegypti. Ae. albopictus, a container-inhabiting mosquito, is an invasive species that occurs over a wide geographic range spanning tropical and temperate latitudes. In this study we examine the effects of a broad range of larval rearing temperatures on CHIKV infection, dissemination, and viral titer in Florida F(1) Ae. albopictus. Adults from larvae reared at 18 degrees C, 24 degrees C, and 32 degrees C differed significantly in size, development time, and CHIKV infection rate. Adult females with the largest body size were produced from the coolest temperature, took the longest to mature, and six times more likely to be infected with CHIKV than females reared at 32 degrees C. There was also a significant effect of rearing temperature on viral dissemination, resulting in an increase in population dissemination at the coolest temperature. This study indicates that climate factors, such as temperature, experienced at the larval stage, can influence the competence of adult females to vector arboviruses.

Environmental and biological factors influencing Culex pipiens quinquefasciatus Say (Diptera: Culicidae) vector competence for Saint Louis encephalitis virus.

Complex interactions between environmental and biological factors influence the susceptibility of Culex pipiens quinquefasciatus to St. Louis encephalitis virus and could affect the epidemiology of virus transmission. Similar interactions could have epidemiologic implications for other vector-virus systems. We conducted an experiment to examine four such factors in combination: mosquito age, extrinsic incubation temperature (EIT), virus dose, and colony. The proportion of mosquitoes with body infections or disseminated infections varied between colonies, and was dependant on age, EIT, and dose. We also show that the probability of a body or leg infection interacted in complex ways between colonies, ages, EITs, and doses. The complex interactive effects of environmental and biological factors must be taken into account for studies of vector competence and epidemiology, especially when laboratory studies are used to generalize to natural transmission dynamics where the extent of variation is largely unknown.

Effect of sequential exposure on infection and dissemination rates for West Nile and St. Louis encephalitis viruses in Culex quinquefasciatus.

West Nile virus has spread rapidly throughout the United States since its introduction in 1999, into some areas that are also endemic for St. Louis encephalitis virus (SLEV). These viruses are in the same antigenic complex within the genus Flavivirus, family Flaviviridae. Further, both viruses are transmitted primarily by Culex spp. mosquitoes and use birds as amplifying hosts. These viruses could contemporaneously coinfect individual vectors wherein changes in mosquito immune responses might occur. To explore this possibility, we evaluated the effect of sequential infection with both West Nile virus and SLEV on the infection and dissemination rates of these viruses in the vector mosquito, Culex quinquefasciatus. Prior exposure to either virus lowered susceptibility to infection with the second virus, and lower dissemination rates of the second virus, compared to controls. Exposure to one virus followed by a second virus resulted in similar infection rates for the first virus to those of controls, but higher SLEV dissemination rates when exposed first to SLEV than in singly SLEV infected controls. While some mosquitoes became infected with both viruses, only one of those viruses disseminated from the midgut into the legs, indicating a midgut infection barrier to secondary infection. Lower infection rates in mosquitoes exposed to both viruses could change transmission patterns when these viruses are present at epizootic levels.

Effects of infectious virus dose and bloodmeal delivery method on susceptibility of Aedes aegypti and Aedes albopictus to chikungunya virus.

Chikungunya virus (CHIKV) is an arbovirus (genus Alphavirus, family Togaviridae) that has recently caused disease outbreaks in the Indian Ocean basin and southern Europe. These outbreaks could be associated with a possible shift in primary vector from Aedes aegypti to Ae. albopictus. To evaluate vector competence differences in possible CHIKV vectors, we evaluated the dose-dependant susceptibility of Florida strains of Ae. albopictus and Ae. aegypti for infection with a La Réunion island strain of CHIKV. Pledget and water-jacketed membrane feeding systems were also evaluated. We show that both Aedes spp. were susceptible to the highest CHIKV doses, whereas only Ae. albopictus developed disseminated infections after exposure to the two lowest doses. Infection rates for both mosquito species were significantly affected by the bloodmeal delivery method used. This information is important in assessing risk of an outbreak of imported CHIKV in the United States, in determining differences in vectorial capacity of these two vector species, and in evaluating arbovirus delivery methods in the laboratory.

Susceptibility of Florida mosquitoes to infection with chikungunya virus.

Chikungunya virus (CHIKV) has caused recent, large epidemics on islands in the Indian Ocean, raising the possibility of more widespread CHIKV epidemics. Historically, CHIKV has been vectored by Aedes aegypti, but these outbreaks likely also involved Ae. albopictus. To examine the potential for an outbreak of CHIKV in Florida, we determined the susceptibility to CHIKV of F1 Ae. aegypti and Ae. albopictus from Florida. In addition, we also evaluated two well-characterized laboratory strains (Rockefeller and Lake Charles) of these species. We determined infection and dissemination rates as well as total body titer of mosquitoes 7 days post-exposure (pe) (Ae. albopictus) and 3, 7, and 10 days pe (Ae. aegypti). All mosquito strains were susceptible to both infection and dissemination, with some variation between strains. Our results suggest Florida would be vulnerable to transmission of CHIKV in urban and rural areas where the two vector species occur.