Negevirus Piura Suppresses Zika Virus Replication in Mosquito Cells.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

Experimental Infection of Mid-Gestation Pregnant Female and Intact Male Sheep with Zika Virus.

Zika virus (ZIKV) is an arbovirus that causes birth defects, persistent male infection, and sexual transmission in humans. The purpose of this study was to continue the development of an ovine ZIKV infection model; thus, two experiments were undertaken. In the first experiment, we built on previous pregnant sheep experiments by developing a mid-gestation model of ZIKV infection. Four pregnant sheep were challenged with ZIKV at 57-64 days gestation; two animals served as controls. After 13-15 days (corresponding with 70-79 days of gestation), one control and two infected animals were euthanized; the remaining animals were euthanized at 20-22 days post-infection (corresponding with 77-86 days of gestation). In the second experiment, six sexually mature, intact, male sheep were challenged with ZIKV and two animals served as controls. Infected animals were serially euthanized on days 2-6 and day 9 post-infection with the goal of isolating ZIKV from the male reproductive tract. In the mid-gestation study, virus was detected in maternal placenta and spleen, and in fetal organs, including the brains, spleens/liver, and umbilicus of infected fetuses. Fetuses from infected animals had visibly misshapen heads and morphometrics revealed significantly smaller head sizes in infected fetuses when compared to controls. Placental pathology was evident in infected dams. In the male experiment, ZIKV was detected in the spleen, liver, testes/epididymides, and accessory sex glands of infected animals. Results from both experiments indicate that mid-gestation ewes can be infected with ZIKV with subsequent disruption of fetal development and that intact male sheep are susceptible to ZIKV infection and viral dissemination and replication occurs in highly vascular tissues (including those of the male reproductive tract).

Experimental Infection of Pregnant Female Sheep with Zika Virus During Early Gestation.

Zika virus (ZIKV) is a vertically and sexually transmissible virus resulting in severe congenital malformation. The goal of this study was to develop an ovine model of ZIKV infection. Between 28-35 days gestation (DG), four pregnant animals were infected with two doses of 6 × 106 PFU of ZIKV; four control animals received PBS. Animals were evaluated for 45 days (D) post-infection (PI) and necropsies were performed. Viral RNA was detected in infected ewe peripheral blood mononuclear cells (PBMC) during the first week PI; however, all fluids and tissues were negative upon culture. Anti-ZIKV IgM (1:400) and neutralizing antibodies were detected in all infected animals. Clinical disease, virus, or ZIKV antibodies were not detected in control ewes. After two weeks PI, fetal loss occurred in two infected animals, and at necropsy, three infected animals had placental petechiation and ecchymosis and one had hydramnion. Fetal morphometrics revealed smaller cranial circumference to crown-rump length ratios (p < 0.001) and relative brain weights (p = 0.038) in fetuses of infected animals compared with control fetuses. Immunophenotyping indicated an increase in B cells (p = 0.012) in infected sheep. Additionally, in vitro experiments using both adult and fetal cell lines demonstrated that ovine cells are highly permissive to ZIKV infection. In conclusion, ZIKV infection of pregnant sheep results in a change in fetal growth and gestational outcomes.

Further Characterization of Molecular Markers in Canine Dirofilaria immitis Infection.

Dirofilaria immitis is a common filarial parasite found in dogs and cats in the Americas, with the pathophysiological consequences of the infection differing somewhat between these 2 host species. Recent research efforts have been focused on determining if the microRNAs (miRNAs) released from adult Dirofilariae have a role as markers for distinguishing the intensity of adult worm infection, as well as determining the presence of new infections. This study expands previous work on 2 nematode miRNAs, miR34 and miR-71, by addressing their ability to discriminate between low and high D. immitis adult worm intensities in dogs. Serum samples were collected from 13 dogs, 8 of which carried known numbers of adult D. immitis at autopsy in their hearts and pulmonary vessels. Three groups of canine sera were created based on D. immitis burden: "control" (0 worms; 5 animals), "low intensity" (10-18 worms; mean ± SD = 12.3 ± 4.4; 4 animals), and "high intensity" (41-72 worms; mean 62.5 ± 15.1; 4 animals) groups. A qPCR analysis was performed on each sample to measure plasma levels of miR-34 and miR-71; however, no significant differences were observed between these groups in terms of levels of miRNAs, so the low- and high-intensity samples were then combined into a single "infected" category and compared to the "non-infected" controls. Copy numbers of both miR-34 and miR-71 were significantly higher in infected compared to uninfected animals ( P = 0.015 and P = 0.027, respectively). The Ct values of expression compared with the adult worm intensity for each miRNA revealed that both miR-34 and miR-71 significantly discriminate between the infected and non-infected groups ( P value < 0.0001 for both). These findings support the contention that miRNA 34 and miRNA 71, which are filarial-specific miRNAs, can both serve as biomarkers for the presence of D. immitis infection in dogs, but at this point they do not appear to reflect the actual intensity of adult parasites present.

Comparison of six commercial antigen kits for detection of Dirofilaria immitis infections in canines with necropsy-confirmed heartworm status.

Patient-side test kits for detecting antigenemia in dogs associated with sexually mature female heartworms (Dirofilaria immitis) have been available for three decades, and these tests are continually updated and improved. To define the sensitivity (Se) and specificity (Sp) of contemporary antigen detection tests against cardiopulmonary D. immitis burden, we evaluated five patient-side kits-Anigen Rapid One Step® (Bio note), SNAP® 4Dx Plus Test Kit (IDEXX), WITNESS® Heartworm Canine Heartworm Antigen Test Kit (Zoetis), VetScan® Canine Heartworm Rapid Test (Abaxis), and Solo Step® CH Canine Heartworm Antigen Test (Heska), and one microplate ELISA (DiroCHEK®; Zoetis), using archived canine sera divided into five subclasses of female worms (0, 1-5, 6-20, 21-40, and >40). The patient-side tests were performed once, side-by-side according to each manufacturer's protocol by personnel blinded to the D. immitis status of each dog. The overall Se and Sp of the patient-side kits was ≥97.5 and =94.0%, respectively. For samples from dogs with 1-5, 6-20, and 21-40 D. immitis, the Se was between 96 and 100%, with a slight increase in Se in dogs with ≥41 worms. The agreement between tests for all subclasses of D. immitis burden was between 99 and 100%. The Se and Sp for the ELISA compared with the necropsy results of dogs was 99 and 96%, respectively. Agreement between each patient-side test and the ELISA was between 97 and 100%. All commercially available tests can give practitioners excellent patient-side information, allowing them to make informed decisions on the need for additional diagnostic work-up before instituting new or continuing D. immitis prophylaxis.