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Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge., Author summary The factors that facilitated the emergence and rapid dissemination of ZIKV in the Americas, and the specific role of strain variation and adaptive evolution in ZIKV transmission, are not fully understood. It has been shown that an amino acid substitution in the NS1 gene acquired prior to the American outbreak confers increased infectivity in Ae. aegypti. Therefore, we hypothesized that variability in the NS1 could contribute to species-specific differences. To investigate the role of the ZIKV NS1 in competence of both Ae. aegypti and Ae. albopictus we utilized reverse genetics to revert to the ancestral NS1 residues and characterized the resultant mutant strains. Reversions increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. Additionally, we assessed the effect of these mutations on NS1 secretion and both virulence and viremia kinetics using a ZIKV mouse model. NS1 secretion, as well as vertebrate morbidity or mortality, were similar among strains, yet strain-specific differences in viremia levels were identified. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Moreover, contrasting these data with previous studies suggests a role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts.

Pathogens possess the ability to adapt and survive in some host species but not in others–an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations., Author summary The evolution of pathogens in response to a host environment often leads to a specificity of these pathogens to survive in particular hosts, known as host tropism. However, the mechanisms of host tropism remain unclear. The Lyme disease (LD) bacterium is an ideal model to study such mechanisms because the causative agent of this disease (bacteria species) are transmitted by ticks and carried by multiple vertebrates, including mammals and birds. We found that different LD bacteria species vary in their ability to survive in mice and quail, and in ticks fed on human or quail blood after transmission. We also showed that such a variation is dependent on whether tick blood meals have functional complement, an innate defense in vertebrate blood. We attributed such species-to-species differences of transmission to the polymorphisms of a bacterial complement inactivating protein, CspA: CspA, by inactivating vertebrate complement in tick blood meals in a species-specific manner, allows bacterial transmission to selective vertebrates. We further demonstrated that this CspA-mediated bacterium species-specific complement evasion arose through convergent evolution, as an evolutionary mechanism to permit such vertebrate-bacteria interaction. The mechanisms defined in this multi-disciplinary work provide new insights into the host-pathogen interactions.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies., Author Summary Since it was first introduced into the United States in 1999, West Nile virus (WNV) has caused significant disease in humans, horses, and other animals. WNV is transmitted to humans and other vertebrate hosts by female mosquitoes as they take a blood meal. Currently, the amount of virus inoculated by mosquitoes while feeding on live hosts is unknown, and accurate estimates are critical so that appropriate challenge doses can be used in vaccine and viral pathogenesis studies. Here, we use a novel technique to determine the dose of WNV inoculated by mosquitoes as they probe and feed on the peripheral tissues of live animals. We found that mosquitoes inoculate high doses of virus into host tissues; these doses are 10 to 1,000 times higher than previous estimates obtained with assays that do not allow mosquitoes to probe or feed naturally. We also found that mosquitoes inoculate low doses of virus directly into the blood while blood feeding. Direct introduction of virus into the blood may alter viral tropism and cause low rates of infection in co-feeding mosquitoes. Our study provides new insights into the transmission of an emerging viral pathogen and the interaction of virus with its mosquito vector and vertebrate host.

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE−/− mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE−/− mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation., Author Summary Several human pathogens express structurally divergent forms of lipid A, the endotoxic portion of lipopolysaccharide (LPS), as a strategy to evade host innate immune detection and establish persistent infection. Expression of modified lipid A species promotes pathogen evasion of host recognition by Toll-like receptor-4 (TLR4) and the non-canonical inflammasome. The Gram-negative oral anaerobe, Porphyromonas gingivalis, expresses lipid A structures that function as TLR4 agonists or antagonists, or are immunologically inert. It is currently unclear how modulation of P. gingivalis lipid A expression contributes to innate immune recognition, survival, and the ability of the pathogen to induce local and systemic inflammation. In this study, we demonstrate that P. gingivalis expression of antagonist lipid A species results in attenuated production of proinflammatory mediators and evasion of non-canonical inflammasome activation, facilitating bacterial survival in the macrophage. Infection of atherosclerosis-prone ApoE−/− mice with this strain resulted in progression of chronic inflammation in the vasculature. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A modifications, supporting distinct mechanisms for induction of local versus systemic inflammation. Our work demonstrates that evasion of immune detection at TLR4 contributes to pathogen persistence and facilitates low-grade chronic inflammation.

The distribution and intensity of transmission of vector-borne pathogens can be strongly influenced by the competence of vectors. Vector competence, in turn, can be influenced by temperature and viral genetics. West Nile virus (WNV) was introduced into the United States of America in 1999 and subsequently spread throughout much of the Americas. Previously, we have shown that a novel genotype of WNV, WN02, first detected in 2001, spread across the US and was more efficient than the introduced genotype, NY99, at infecting, disseminating, and being transmitted by Culex mosquitoes. In the current study, we determined the relationship between temperature and time since feeding on the probability of transmitting each genotype of WNV. We found that the advantage of the WN02 genotype increases with the product of time and temperature. Thus, warmer temperatures would have facilitated the invasion of the WN02 genotype. In addition, we found that transmission of WNV accelerated sharply with increasing temperature, T, (best fit by a function of T4) showing that traditional degree-day models underestimate the impact of temperature on WNV transmission. This laboratory study suggests that both viral evolution and temperature help shape the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on WNV transmission., Author Summary West Nile virus (WNV) was introduced into New York in 1999 and subsequently expanded its range to include much of North, Central, and South America. Previously, we have shown that a new strain of WNV (referred to as WN02) that was first detected in 2001 and subsequently spread across North America was more efficient at infecting and being transmitted by Culex mosquitoes than the strain that was originally introduced (referred to as NY99). In the current study, we determined how temperature and time since feeding on infected blood affected the probability that mosquitoes would transmit these two strains of WNV. We found that the advantage of the WN02 strain over the NY99 strain increased with both temperature and time. Thus, warmer temperatures would have facilitated the invasion of the WN02 strain. In addition, we found that transmission of both strains of WNV accelerated sharply with increasing temperature, such that small increases in temperature had relatively large effects on transmission. This laboratory study suggests that both viral evolution and temperature influence the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on virus transmission.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses., Author Summary West Nile virus (WNV) is the most geographically widespread arthropod-borne virus (arbovirus) in the world. Like most arboviruses, WNV is a RNA virus which is highly mutable and exists in nature as genetically diverse mutant swarms. Although many recent studies have investigated the relationship between virus mutation rate and viral fitness, this had not previously been determined for WNV or other flaviviruses. We identified WNV mutations associated with variation in mutation rate using cell culture passage in the presence of a mutagen and engineered these mutations into an infectious WNV clone in order to investigate the causes and consequences of altered fidelity. Our results demonstrate that interactions among proteins which comprise the WNV replication complex can significantly alter both the extent and types of mutations that occur. In addition, we show that both increasing and decreasing WNV fidelity has host-specific effects on replication in cell culture and is associated with nearly complete ablation of WNV infection in mosquito vectors. These results have significant implications for our understanding of arbovirus evolution, replication complex function and arboviral fitness in mosquitoes, and identify important targets to study the determinants and mechanisms of vector competence and arbovirus fidelity.