Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection.

Zika virus (ZIKV) is a mosquito-borne member of the genus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was> 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30–90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0–4.2°C), respiration rate (+56–128%), activity (-15-76% daytime and +5–22% nighttime), heart rate (+67–190%), systolic (+44–67%) and diastolic blood pressure (+45–80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (12–24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106–140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.

Mosquito-borne and sexual transmission of Zika virus (ZIKV), a TORCH pathogen, recently initiated a series of large epidemics throughout the Tropics. Animal models are necessary to determine transmission risk and study pathogenesis, as well screen antivirals and vaccine candidates. In this study, we modeled mosquito and sexual transmission of ZIKV in the African green monkey (AGM). Following subcutaneous, intravaginal or intrarectal inoculation of AGMs with ZIKV, we determined the transmission potential and infection dynamics of the virus. AGMs inoculated by all three transmission routes exhibited viremia and viral shedding followed by strong virus neutralizing antibody responses, in the absence of clinical illness. All four of the subcutaneously inoculated AGMs became infected (mean peak viremia: 2.9 log 10 PFU/mL, mean duration: 4.3 days) and vRNA was detected in their oral swabs, with infectious virus being detected in a subset of these specimens. Although all four of the intravaginally inoculated AGMs developed virus neutralizing antibody responses, only three had detectable viremia (mean peak viremia: 4.0 log 10 PFU/mL, mean duration: 3.0 days). These three AGMs also had vRNA and infectious virus detected in both oral and vaginal swabs. Two of the four intrarectally inoculated AGMs became infected (mean peak viremia: 3.8 log 10 PFU/mL, mean duration: 3.5 days). vRNA was detected in oral swabs collected from both of these infected AGMs, and infectious virus was detected in an oral swab from one of these AGMs. Notably, vRNA and infectious virus were detected in vaginal swabs collected from the infected female AGM (peak viral load: 7.5 log 10 copies/mL, peak titer: 3.8 log 10 PFU/mL, range of detection: 5-21 days post infection). Abnormal clinical chemistry and hematology results were detected and acute lymphadenopathy was observed in some AGMs. Infection dynamics in all three AGM ZIKV models are similar to those reported in the PLOS NEGLECTED TROPICAL DISEASES

To evaluate potential immunocompetent small animal models of Zika virus (ZIKV) infection, we inoculated Syrian golden hamsters (subcutaneously or intraperitoneally) and strain 13 guinea pigs (intraperitoneally) with Senegalese ZIKV strain ArD 41525 or Philippines ZIKV strain CPC-0740. We did not detect viremia in hamsters inoculated subcutaneously with either virus strain, although some hamsters developed virus neutralizing antibodies. However, we detected statistically significant higher viremias ( = 0.0285) and a higher median neutralization titer ( = 0.0163) in hamsters inoculated intraperitoneally with strain ArD 41525 compared with strain CPC-0740. Furthermore, some hamsters inoculated with strain ArD 41525 displayed mild signs of disease. By contrast, strain 13 guinea pigs inoculated intraperitoneally with either strain did not have detectable viremias and less than half developed virus neutralizing antibodies. Our results support the use of the Syrian golden hamster intraperitoneal model to explore phenotypic variation between ZIKV strains.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2 °C), respiration rate (+56-128%), activity (+15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ∼99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ∼106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.Author SummaryIn North America, EEEV causes the most severe mosquito-borne disease in humans highlighted by fatal encephalitis and permeant debilitating neurological sequelae in survivors. The first confirmed human cases were reported more than 80 years ago and since then multiple sporadic outbreaks have occurred including one of the largest in 2019. Unfortunately, most human infections are diagnosed at the on-set of severe neurological symptoms and consequently a detailed disease course in humans is lacking. This gap in knowledge is a significant obstacle in the development of appropriate animal models to evaluate countermeasures. Here, we performed a cutting-edge study by utilizing a new telemetry technology to understand the course of EEEV infection in a susceptible macaque model by measuring multiple physiological parameters relevant to human disease. Our study demonstrates that the infection rapidly produces considerable alterations in many critical parameters including the electrical activity of the heart and the brain leading to severe disease. The study also highlights the extraordinary potential of new telemetry technology to develop the next generation of animal models in order to comprehensively investigate pathogenesis as well as evaluate countermeasures to treat and/or prevent EEEV disease.

Laboratory animals are commonly anesthetized to prevent pain and distress and to provide safe handling. Anesthesia procedures are well-developed for common laboratory mammals, but not as well established in reptiles. We assessed the performance of intramuscularly injected tiletamine (dissociative anesthetic) and zolazepam (benzodiazepine sedative) in fixed combination (2 mg/kg and 3 mg/kg) in comparison to 2 mg/kg of midazolam (benzodiazepine sedative) in ball pythons (Python regius). We measured heart and respiratory rates and quantified induction parameters (i.e., time to loss of righting reflex, time to loss of withdrawal reflex) and recovery parameters (i.e., time to regain righting reflex, withdrawal reflex, normal behavior). Mild decreases in heart and respiratory rates (median decrease of <10 beats per minute and <5 breaths per minute) were observed for most time points among all three anesthetic dose groups. No statistically significant difference between the median time to loss of righting reflex was observed among animals of any group (p = 0.783). However, the withdrawal reflex was lost in all snakes receiving 3mg/kg of tiletamine+zolazepam but not in all animals of the other two groups (p = 0.0004). In addition, the time for animals to regain the righting reflex and resume normal behavior was longer in the drug combination dose groups compared to the midazolam group (p = 0.0055). Our results indicate that midazolam is an adequate sedative for ball pythons but does not suffice to achieve reliable immobilization or anesthesia, whereas tiletamine+zolazepam achieves short-term anesthesia in a dose-dependent manner.