Background: Chagas disease is one of the world's most neglected tropical diseases, infecting over six million people across the Americas. The hemoparasite Trypanosoma cruzi is the etiological agent for the disease, circulating in domestic, peridomestic, and sylvatic transmission cycles that are maintained by triatomine vectors and a diversity of wild and synanthropic hosts. Public health and wildlife management interventions targeting the interruption of T. cruzi transmission rely on an understanding of the dynamics driving the ecology of this zoonotic pathogen. One wildlife host that purportedly plays a role in the transmission of Chagas disease within the southern United States is the striped skunk (Mephitis mephitis), although infection prevalence in this species is poorly understood. Materials and Methods: To this end, we conducted a PCR-based surveillance of T. cruzi in 235 wild skunks, representing 4 species, across 76 counties and 10 ecoregions in Texas, United States, along with an evaluation of risk factors associated with the infection. Results: We recovered an overall T. cruzi prevalence of 17.9% for all mephitid taxa aggregated, ranging between 6.7% for plains spotted skunks (Spilogale putorius interrupta) and 42.9% for western spotted skunks (Spilogale gracilis). We report the first cases of T. cruzi infection in plains spotted and American hog-nosed skunks (Conepatus leuconotus), of important note for conservation medicine since populations of both species are declining within Texas. Although not statistically significant, we also detected trends for juveniles to exhibit greater infection risk than adults and for differential sex biases in T. cruzi prevalence between taxa, which align with variations in species-specific seasonal activity patterns. No geographic or taxonomic risk factors were identified. Conclusion: Our study contributed key data for population viability analyses and epidemiologic models in addition to providing a baseline for future T. cruzi surveillance among skunks and other wildlife species.

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

To investigate possible cardiac manifestations of Chagas disease, we tested 97 Latinx patients with nonischemic cardiomyopathy in Houston, Texas, USA, for Trypanosoma cruzi infection. We noted a high prevalence of underdiagnosed infection and discrepant results in clinical diagnostic assays. Latinx cardiac patients in the United States would benefit from laboratory screening for T. cruzi infection.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

West Nile virus (WNV) is a widespread and devastating disease, especially in those who develop neuroinvasive disease. A growing body of evidence describes sequelae years after infection, including neurological complications and chronic kidney disease (CKD). Eighty-nine out of 373 WNV-positive cases were followed for approximately two years and compared to 127 WNV-negative controls with and without CKD. Adjusted risk ratios (aRRs) were calculated via a log binomial regression to determine the impact of WNV exposure and other possible confounders on the likelihood of developing CKD. Cytokine profiles of WNV patients and controls were evaluated to characterize differences and describe potential underlying pathophysiological mechanisms. The associated risk for developing CKD was significantly associated with history of WNV infection (aRR = 1.91, 95% CI 1.13–3.25). Additionally, five distinct cytokines were found to be significantly associated with WNV infection (eotaxin, IL-8, IL-12p70, IP-10, and TNFα) after the p-value was adjusted to <0.0019 due to the Bonferroni correction. These data support that WNV infection is an independent risk factor for CKD, even after accounting for confounding comorbidities. WNV participants who developed CKD had high activity of proinflammatory markers, indicating underlying inflammatory disease. This study provides new insights into CKD resultant of WNV infection.

In the Trans-Pecos region of Texas, reports of domestic triatomine bites were common (67%), with 36% of residentially collected triatomines positive for Trypanosoma cruzi. Despite the transmission potential, no human infections were detected. Collected Triatoma rubida species were themselves frequently parasitized with mites.

Venezuelan equine encephalitis virus (VEEV) is a re-emerging virus of human, agriculture, and bioweapon threat importance. No FDA-approved treatment is available to combat Venezuelan equine encephalitis in humans, prompting the need to create a vaccine that is safe, efficacious, and cannot be replicated in the mosquito vector. Here we describe the use of a serotype ID VEEV (ZPC-738) vaccine with an internal ribosome entry site (IRES) to alter gene expression patterns. This ZPC/IRES vaccine was genetically engineered in two ways based on the position of the IRES insertion to create a vaccine that is safe and efficacious. After a single dose, both versions of the ZPC/IRES vaccine elicited neutralizing antibody responses in mice and non-human primates after a single dose, with more robust responses produced by version 2. Further, all mice and primates were protected from viremia following VEEV challenge. These vaccines were also safer in neonatal mice than the current investigational new drug vaccine, TC-83. These results show that IRES-based attenuation of alphavirus genomes consistently produce promising vaccine candidates, with VEEV/IRES version 2 showing promise for further development.

Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA) protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus.

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.

Maternal ZIKV infection periconception results in CZS in a significant minority of cases. Our recent data demonstrated that ZIKA infection also results in early pregnancy loss in several non-human primate models (PMID 29967348, 29717225). However, estimates of rates of ZIKV vertical transmission and associated pregnancy loss vary in human populations, and are likely dependent on host, viral and other factors. Commensal bacterial microbes, for example, may be important mediators of immunity and viral infection. Here we hypothesized that host bacteria would mediate ZIKV vertical transmission and associated pregnancy loss. Timed-pregnant Swiss-Webster mice, that were either conventional or bred and housed in the absence of bacteria (gnotobiotic germ-free; Fig. A), were inoculated with 1x104PFU of a contemporaneous first passage ZIKV or a mock injection (M) on embryonic days 4 through 7. A group was also treated with anti-IFNI1 antibody + ZIKV as a positive control. Caesarean deliveries of pups and placentae were performed at day 18.5. Animals were assessed for viral levels, as well as fetal demise and growth restriction. Mothers were asymptomatic for ZIKV infection based on daily monitoring. Overall, germ-free (GF) mice had higher rates of vertical transmission as measured by fetal growth restriction (1.0 g vs 1.2 g, GF vs conventional p<0.01), and a significantly higher rate intrauterine demise on day 18.5 (OR 8.7 c2p=0.01) (Fig. B). Among ZIKA infected gestations, placentae were positive in 82% of demised fetus' compared to 52% of live pups (p=0.04) (Fig. C). 11 days after infection, viral RNA was recovered from maternal spleen and uterus, and was highest by an order of magnitude in fetal placental tissue overall (7.9x103, 1.9x103vs 6.1x104, respectively, p<0.001). Of particular interest, ZIKV levels among GF mice when compared with conventional were higher in spleen (2.1 x 104vs 8.7 x103 RNA copies/g; p=0.04) and uterus (1.0 x104 vs 6.1 x 102 RNA copies/g, p=0.04) (Fig D). Overall, 25% of conventional dams were infected compared to 78% of GF (c2, p=0.03). We tested the novel hypothesis that maternal antiviral immunity may be stimulated or augmented by the presence of commensal bacteria in pregnancy. We have shown for the first time that germ-free dams and their pups are more susceptible to ZIKV infection and CZS, suggesting the mitigating potential of commensal bacteria in congenital viral infections.

Eleven triatomine species, the vector for Chagas disease, are endemic in the southern U.S. While traditionally thought to only occur in rural habitats and sylvatic transmission cycles, recent studies provide compounding evidence that triatomines could exist in urban habitats and domestic transmission cycles in Texas. We conducted a study of active and passive surveillance techniques over 3 years (2016-2018) in the City of Houston, Harris County, Texas to determine the presence of triatomines in this metroplex. Active surveillance methods uncovered Triatoma sanguisuga nymphs from two locations in downtown Houston city parks. We also documented the first Trypanosoma cruzi positive kissing bug collected in an urban environment of Harris County, Texas. Our findings provide evidence that triatomines can be found in heavily populated U.S. urban environments, and warrant public health support for expanded triatomine and Chagas disease surveillance in city settings.

West Nile virus (WNV) is an arbovirus with important public health implications globally. This study characterizes a viral isolate, 2004Hou3, in comparison with the NY99 strain from the original WNV outbreak in New York, USA. NextGen sequencing was used to compare the viral isolates genetically, while wild-type C57/BL6 mice were used to compare pathogenicity and viral persistence. Significant differences in survival and clinical presentations were noted, with minor genetic variations between the two strains potentially offering an explanation. One notable difference is that 5 of 35 mice infected with the 2004Hou3 strain developed hind limb flaccid paralysis, suggesting its possible use as a small animal pathogenesis model for this clinical characteristic often observed in human WN neuroinvasive disease patients but not reported in other animal models of infection. Overall, this study suggests that 2004Hou3 is a less pathogenic strain with potential for use in long-term outcome studies using small animal models.

Recent findings reveal that commensal microbiota may play a role in protection against viral infections. For example, bacteria isolated from the human stomach confer resistance against rotavirus infection in mice. Vertical transmission of ZIKV has been shown cause IUGR in mice. Here we sought to determine the contribution of commensal bacteria in protecting against ZIKV infection, vertical transmission, and IUGR. Current pandemic ZIKV was isolated from a traveler returning from Central America and propagated in Vero cells (ZIKV HN16). Pregnant germ-free (GF) and conventional (CONV) Swiss-Webster mice were intravenously inoculated with 104 PFU of second passage ZIKV (n=11) or a mock (n=10) injection on embryonic day 6 (e6), then daily until day e9 (Figure 1). Mice (n=2) were given anti-IFNI1 ab prior to and after infection as positive infection controls. On e18, Cesareans were performed and mice assessed for intrauterine reabsorption and demise as well as embryonic pup weight. Tissues were assessed for ZIKV infection by TaqMan viral qPCR assay. Inoculated mice were asymptomatic based on daily assessment of mobility and paralysis. Overall, GF-ZIKV pups had a greater rate of in utero demise with ZIKV infection (p< 0.01), while there was no difference for conventional controls (Fig 2A). In GF-ZIKV dams, ZIKV was detected in 3 of 4 spleens assessed, but only detected in 1 of 4 spleens from GF-ZIKV mice, and with 100-fold less viral load (B). The uterus was positive for ZIKV in 75% (3/4) GF-ZIKV dams in comparison to 50% (2/4) CONV-ZIKV dams, also with 100-fold less viral load (B). Of the mice positive for ZIKV in the uterus, 3 of 4 had associated in utero demise, while all pregnancies with undetectable levels of ZIKV in the uterus had no incidences of in utero demise (Fig A/B?). Pups of Zika infected conventional mothers showed no difference in fetal weight compared to mock, while germ free mothers were 25% smaller by weight (Fig C) (p<0.0001), indicating protection of the native flora. The findings demonstrate that commensal bacteria confer protection against fetal/placental ZIKV infection and transmission either through direct host defense or mediated through adaptive immunity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

We instituted active surveillance among febrile patients presenting to the largest Houston-area pediatric emergency department to identify acute infections of dengue virus (DENV), West Nile virus (WNV), and chikungunya virus (CHIKV). In 2014, 1,063 children were enrolled, and 1,015 (95%) had blood and/or cerebrospinal fluid specimens available for DENV, WNV, and CHIKV testing. Almost half (49%) reported recent mosquito bites, and 6% (N = 60) reported either recent international travel or contact with an international traveler. None were positive for acute WNV; three had false-positive CHIKV results; and two had evidence of DENV. One DENV-positive case was an acute infection associated with international travel, whereas the other was identified as a potential secondary acute infection, also likely travel-associated. Neither of the DENV-positive cases were clinically recognized, highlighting the need for education and awareness. Health-care professionals should consider the possibility of arboviral disease among children who have traveled to or from endemic areas.

Recent evidence has demonstrated that babies born with congenital Zika syndrome (CZS) can test negative due to prenatal clearance of the virus. Conversely, among latter 2nd and 3rd trimester exposures, clinical evidence of CZS may not appear until after deliver. This is modeled in mice, where we have previously demonstrated only 4% of the offspring of ZIKV inoculated dams are positive for ZIKV despite widespread postnatal growth restriction. We therefore hypothesized that mice of these pregnancies would show lasting changes to regulatory genes of innate immunity such as interferon stimulated genes as evidence of the longer term effects of congenital ZIKV exposure and clearance. Timed, pregnant Swiss-Webster mice immunosuppressed with anti-interferon alpha receptor were inoculated with ZIKV HN16 or control on embryonic day 8 (e8). Brain tissue of offspring was assessed at either postnatal day 1 (P1) or 6 weeks of age for the following interferon stimulated gene expression: Isg15, Ifitm3, Oas1b, SOCS1, or SOCS3. PCR was performed via gel electrophoresis as well as qPCR via SYBR Green using the delta delta CT methodology. Mice in the mid-gestation ZIKV infected group had significantly higher rates of growth restriction compared to control (Figure 1A) (p=0.007). Within 12 ZIKV-exposed offspring brain tissue, there was decreased expression of Oas1b at birth (P1) by 22.4% (p=0.003) and SOCS1 appeared to be upregulated by 38% (p=0.055) compared to control (Figure 1B). However, at 6 weeks of age, Ifitm3 expression was 33.6% increased compared to control (p=0.029), Oas1b was trending upwards by 42% (p=0.094) and SOCS3 by 45% (p=0.057) but did not reach significance (Figure 1C). We have shown, for the first time, the persistence of dysregulation of interferon stimulated genes gene expression among congenital ZIKV-exposed, but not affected, offspring through adolescence in a murine model. IFN gene regulation changes preceded the development of clinically evident disease. Given that these same gene changes are known to be persistently altered in other congenital viral syndromes with neuropathology (i.e., HIV), these findings suggest that ISGs are active throughout development and into adolescence. We speculate that our findings may signal that ZIKV exposure during gestation, even in the absence of CZS, may have lasting neuropathic effects among offspring.