Seroprevalence of Zika virus in wild African green monkeys and baboons

ABSTRACT Zika virus (ZIKV) has recently spread through the Americas and has been associated with a range of health effects, including birth defects in children born to women infected during pregnancy. Although the natural reservoir of ZIKV remains poorly defined, the virus was first identified in a captive “sentinel” macaque monkey in Africa in 1947. However, the virus has not been reported in humans or nonhuman primates (NHPs) in Africa outside Gabon in over a decade. Here, we examine ZIKV infection in 239 wild baboons and African green monkeys from South Africa, the Gambia, Tanzania, and Zambia using combinations of unbiased deep sequencing, quantitative reverse transcription-PCR (qRT-PCR), and an antibody capture assay that we optimized using serum collected from captive macaque monkeys exposed to ZIKV, dengue virus, and yellow fever virus. While we did not find evidence of active ZIKV infection in wild NHPs in Africa, we found variable ZIKV seropositivity of up to 16% in some of the NHP populations sampled. We anticipate that these results and the methodology described within will help in continued efforts to determine the prevalence, natural reservoir, and transmission dynamics of ZIKV in Africa and elsewhere. IMPORTANCE Zika virus (ZIKV) is a mosquito-borne virus originally discovered in a captive monkey living in the Zika Forest of Uganda, Africa, in 1947. Recently, an outbreak in South America has shown that ZIKV infection can cause myriad health effects, including birth defects in the children of women infected during pregnancy. Here, we sought to investigate ZIKV infection in wild African primates to better understand its emergence and spread, looking for evidence of active or prior infection. Our results suggest that up to 16% of some populations of nonhuman primate were, at some point, exposed to ZIKV. We anticipate that this study will be useful for future studies that examine the spread of infections from wild animals to humans in general and those studying ZIKV in primates in particular. Podcast: A podcast concerning this article is available

Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment

Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of influenza infection and the associated host innate immune response

Zika virus persistence in the male macaque reproductive tract.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility

Seroprevalence of Zika Virus in Wild African Green Monkeys and Baboons.

Zika virus (ZIKV) has recently spread through the Americas and has been associated with a range of health effects, including birth defects in children born to women infected during pregnancy. Although the natural reservoir of ZIKV remains poorly defined, the virus was first identified in a captive "sentinel" macaque monkey in Africa in 1947. However, the virus has not been reported in humans or nonhuman primates (NHPs) in Africa outside Gabon in over a decade. Here, we examine ZIKV infection in 239 wild baboons and African green monkeys from South Africa, the Gambia, Tanzania, and Zambia using combinations of unbiased deep sequencing, quantitative reverse transcription-PCR (qRT-PCR), and an antibody capture assay that we optimized using serum collected from captive macaque monkeys exposed to ZIKV, dengue virus, and yellow fever virus. While we did not find evidence of active ZIKV infection in wild NHPs in Africa, we found variable ZIKV seropositivity of up to 16% in some of the NHP populations sampled. We anticipate that these results and the methodology described within will help in continued efforts to determine the prevalence, natural reservoir, and transmission dynamics of ZIKV in Africa and elsewhere. IMPORTANCE Zika virus (ZIKV) is a mosquito-borne virus originally discovered in a captive monkey living in the Zika Forest of Uganda, Africa, in 1947. Recently, an outbreak in South America has shown that ZIKV infection can cause myriad health effects, including birth defects in the children of women infected during pregnancy. Here, we sought to investigate ZIKV infection in wild African primates to better understand its emergence and spread, looking for evidence of active or prior infection. Our results suggest that up to 16% of some populations of nonhuman primate were, at some point, exposed to ZIKV. We anticipate that this study will be useful for future studies that examine the spread of infections from wild animals to humans in general and those studying ZIKV in primates in particular

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens

79: Modeling Zika Virus Tissue Tropism in Rhesus Macaques to Define the Risk of Donor-derived Transmission

Abstract Background Almost 115,000 people in the United States are currently on a transplant waitlist, which vastly exceeds the number of organ donors every year. This discrepancy emphasizes the need for retention of all possible donors. Those who have recently traveled to an area with an active outbreak of Zika virus (ZIKV) are often disqualified as a donor because immunosuppressed recipients would be at risk of a donor-derived ZIKV infection. Therefore, we define ZIKV tissue tropism and the risk of donor-derived transmission. Methods We subcutaneously inoculated 15 Indian-origin rhesus macaques (RM) with a Puerto Rican isolate of ZIKV (PRVABC59). All RMs were inoculated in mid to early gestation. We inoculated during pregnancy because plasma viremia is typically prolonged in pregnancy and we wanted to model tissue tropism for donor-derived transmission in the worst scenario of prolonged viremia. At 30, 65, and 105 days post-infection (dpi), the animals were euthanized and comprehensive necropsies were performed, which evaluated a minimum of 60 tissues per animal. ZIKV RNA was quantified in tissues via qRT-PCR. Results Plasma viremia duration was >10 days in 13 of 15 RMs. ZIKV RNA was most commonly detected in lymph nodes, with 19/45 lymph nodes that were vRNA positive in 5 RMs at 30 dpi. There were 15/45 vRNA positive lymph nodes at 60 dpi and 8/38 at 105 dpi. Reproductive and maternal fetal-interface (MFI) tissues were the second most commonly positive tissues. Twenty-five MFI tissues, including the amniotic/chorionic membrane, decidua, placenta, uterus, and placental bed, were positive, with 10/53 positive at 30 dpi, 14/24 positive at 60 dpi and 1/47 positive at 105 dpi. Other vRNA positive tissues included the primary bronchus, femoral vein, kidney, thyroid, lung, colon, mammary gland, pericardium, hand nerve, and sciatic nerve in 1–2 RMs at one of the three timepoints. Conclusions We found ZIKV RNA most frequently within lymph nodes. Lymph nodes are included in lung and small bowel transplants, indicating that these transplants could pose a risk of donor-derived ZIKV transmission. Virus detection within other commonly transplanted tissues, such as the kidney and blood vessels was much less common. We did not determine what fraction of vRNA comes from replication-competent virus in each tissue; some tissues with vRNA might not contain virions that could initiate new infections. Donor-derived Zika virus transmission from other commonly transplanted organs, such as the liver, seems unlikely since no viral RNA was detected in this organ

20: Modeling Zika virus tissue tropism in rhesus macaques to define the risk of donor derived transmission

Abstract Background Almost 115,000 people in the United States are currently on a transplant waitlist, which vastly exceeds the number of organ donors every year. This discrepancy emphasizes the need for retention of all possible donors. Those who have recently traveled to an area with an active outbreak of Zika virus (ZIKV) are often disqualified as a donor because immunosuppressed recipients would be at risk of a donor-derived ZIKV infection. Therefore, we define ZIKV tissue tropism and the risk of donor derived transmission. Methods We subcutaneously inoculated 15 Indian-origin rhesus macaques (RM) with a Puerto Rican isolate of ZIKV (PRVABC59). All RMs were inoculated in mid to early gestation.We inoculated during pregnancy because plasma viremia is typically prolonged in pregnancy and we wanted to model tissue tropism for donor derived transmission in the worst scenario of prolonged viremia. At 30, 65, and 105 days post-infection (dpi), the animals were euthanized and comprehensive necropsies were performed, which evaluated a minimum of 60 tissues per animal. ZIKV RNA was quantified in tissues via qRT-PCR. Results Plasma viremia duration was >10 days in 13 of 15 RMs. ZIKV RNA was most commonly detected in lymph nodes, with 19/45 lymph nodes that were vRNA positive in 5 RMs at 30 dpi. There were 15/45 vRNA positive lymph nodes at 60 dpi and 8/38 at 105 dpi. Reproductive and maternal fetal-interface (MFI) tissues were the second most commonly positive tissues. Twenty-five MFI tissues, including the amniotic/chorionic membrane, decidua, placenta, uterus, and placental bed, were positive, with 10/53 positive at 30 dpi, 14/24 positive at 60 dpi and 1/47 positive at 105 dpi. Other vRNA positive tissues included the primary bronchus, femoral vein, kidney, thyroid, lung, colon, mammary gland, pericardium, hand nerve, and sciatic nerve in 1–2 RMs at one of the three timepoints. Conclusions We found ZIKV RNA most frequently within lymph nodes. Lymph nodes are included in lung and small bowel transplants, indicating that these transplants could pose a risk of donor-derived ZIKV transmission. Virus detection within other commonly transplanted tissues, such as the kidney and blood vessels was much less common. We did not determine what fraction of vRNA comes from replication-competent virus in each tissue; some tissues with vRNA might not contain virions that could initiate new infections. Donor-derived Zika virus transmission from other commonly transplanted organs, such as liver, seems unlikely since no viral RNA was detected in this organ

Primary infection with dengue or Zika virus does not affect the severity of heterologous secondary infection in macaques.

Zika virus (ZIKV) and dengue virus (DENV) are genetically and antigenically related flaviviruses that now co-circulate in much of the tropical and subtropical world. The rapid emergence of ZIKV in the Americas in 2015 and 2016, and its recent associations with Guillain-Barré syndrome, birth defects, and fetal loss have led to the hypothesis that DENV infection induces cross-reactive antibodies that influence the severity of secondary ZIKV infections. It has also been proposed that pre-existing ZIKV immunity could affect DENV pathogenesis. We examined outcomes of secondary ZIKV infections in three rhesus and fifteen cynomolgus macaques, as well as secondary DENV-2 infections in three additional rhesus macaques up to a year post-primary ZIKV infection. Although cross-binding antibodies were detected prior to secondary infection for all animals and cross-neutralizing antibodies were detected for some animals, previous DENV or ZIKV infection had no apparent effect on the clinical course of heterotypic secondary infections in these animals. All animals had asymptomatic infections and, when compared to controls, did not have significantly perturbed hematological parameters. Rhesus macaques infected with DENV-2 approximately one year after primary ZIKV infection had higher vRNA loads in plasma when compared with serum vRNA loads from ZIKV-naive animals infected with DENV-2, but a differential effect of sample type could not be ruled out. In cynomolgus macaques, the serotype of primary DENV infection did not affect the outcome of secondary ZIKV infection

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques.

BACKGROUND:Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV. METHODOLOGY/PRINCIPAL FINDINGS:Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form. CONCLUSIONS/SIGNIFICANCE:An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains