Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

Serological Evidence of Zika Virus Infections in Sudan.

Little is known about the frequency of Zika virus (ZIKV) infections in Sudan. The aim of this study was to obtain data on the prevalence of ZIKV infections and the immunity of the population in the country. To this end, 198 sera obtained between December 2012 and January 2013 in different regions in Sudan were examined for neutralizing antibodies against ZIKV, dengue virus (DENV), and yellow fever virus (YFV). The sera were non-randomly selected. The neutralization titers were compared with each other and with the WHO 1st International Standard for anti-Asian lineage Zika virus antibody. Twenty-six sera neutralized ZIKV. One-third of these sera had higher neutralization titers against ZIKV than against DENV-2 and -3. Two sera showed higher neutralization titers than the WHO standard for ZIKV antibodies. These data suggest occasional ZIKV infections in Sudan. The low percentage of sera in this cohort that neutralized ZIKV indicates that, in the study period, the population was susceptible to ZIKV infection.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

Sustained chronic inflammation and altered childhood vaccine responses in children exposed to Zika virus.

BACKGROUND: Congenital Zika virus (ZIKV) infection leads to severe newborn abnormalities, but its long-term impact on childhood immunity is not well understood. This study aims to investigate the serum proteomics in children exposed to ZIKV during pregnancy to understand potential immunological consequences during early childhood. METHODS: The study included ZIKV-exposed infants (ZEI) at birth (n = 42) and children exposed to ZIKV (ZEC) at two years of age (n = 20) exposed to ZIKV during pregnancy, as well as healthy controls. Serum proteomic analysis was performed on these groups to assess inflammation and immune profiles. Additionally, antibody titres against two common childhood vaccines, DTaP and MMR, were measured in healthy controls (n = 50) and ZEC (n = 92) to evaluate vaccine-induced immunity. FINDINGS: Results showed elevated inflammation in ZEI with birth abnormalities. Among ZEC, despite most having normal clinical outcomes at two years, their serum proteomics indicated a bias towards Th1-mediated immune responses. Notably, ZEC displayed reduced anti-Diphtheria toxin and anti-Clostridium tetani IgG levels against DTaP and MMR vaccines. They also exhibited lower antibody titres particularly against Th2-biased DTaP vaccines, but not Th1-biased MMR vaccines. INTERPRETATION: In conclusion, the study highlights the long-term immunological consequences of congenital ZIKV exposure. Heightened inflammation was observed in ZEI with abnormalities at birth, while ZEC maintained a chronic Th1-biased immune profile. The impaired response to Th2-biased vaccines raises concerns about lasting effects of ZIKV exposure on immune responses. Consequently, there is a need for continued longitudinal clinical monitoring to identify potential immune-related complications arising from prenatal exposure to ZIKV. FUNDING: This work was partially funded by the National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Dental and Craniofacial Research (NIDCR).

Seroprevalence of Arboviruses in a Malaria Hyperendemic Area in Southern Mali.

Diagnostics for febrile illnesses other than malaria are not readily available in rural sub-Saharan Africa. This study assessed exposure to three mosquito-borne arboviruses-dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV)-in southern Mali. Seroprevalence for DENV, CHIKV, and ZIKV was analyzed by detection of IgG antibodies and determined to be 77.2%, 31.2%, and 25.8%, respectively. Among study participants, 11.3% were IgG-positive for all three arboviruses. DENV had the highest seroprevalence rate at all sites; the highest seroprevalence of CHIKV and ZIKV was observed in Bamba. The seroprevalence for all three arboviruses increased with age, and the highest seroprevalence was observed among adults older than 50 years. The prevalence of Plasmodium spp. in the cohort was analyzed by microscopy and determined to be 44.5% (N = 600) with Plasmodium falciparum representing 95.1% of all infections. This study demonstrates the co-circulation of arboviruses in a region hyperendemic for malaria and highlights the needs for arbovirus diagnostics in rural sub-Saharan Africa.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Dengue Virus and Zika Virus Seroprevalence in the South Pacific Populations of the Cook Islands and Vanuatu.

Arboviral diseases are serious threats to global health with increasing prevalence and potentially severe complications. Significant arthropod-borne viruses are the dengue viruses (DENV 1-4), the Zika virus (ZIKV), and the chikungunya virus (CHIKV). Among the areas most affected is the South Pacific Region (SPR). Here, arboviruses not only cause a high local burden of disease, but the region has also proven to contribute to their global spread. Outpatient serum samples collected between 08/2016 and 04/2017 on three islands of the island states of Vanuatu and the Cook Islands were tested for anti-DENV- and anti-ZIKV-specific antibodies (IgG) using enzyme-linked immunosorbent assays (ELISA). ELISA test results showed 89% of all test sera from the Cook Islands and 85% of the Vanuatu samples to be positive for anti-DENV-specific antibodies. Anti-ZIKV antibodies were identified in 66% and 52%, respectively, of the test populations. Statistically significant differences in standardized immunity levels were found only at the intranational level. Our results show that in both the Cook Islands and Vanuatu, residents were exposed to significant Flavivirus transmission. Compared to other seroprevalence studies, the marked difference between ZIKV immunity levels and previously published CHIKV seroprevalence rates in our study populations is surprising. We propose the timing of ZIKV and CHIKV emergence in relation to recurrent DENV outbreaks and the impact of seasonality as explanatory external factors for this observation. Our data add to the knowledge of arboviral epidemics in the SPR and contribute to a better understanding of virus spread, including external conditions with potential influence on outbreak dynamics. These data may support preventive and rapid response measures in the affected areas, travel-related risk assessment, and infection identification in locals and returning travelers.

Harmonization of Zika serological assays and comparative evaluation of two commercial ZIKA IgG ELISA kits.

The interpretation for Zika virus serology results is challenging due to high antibody cross reactivity with other flaviviruses. This limits availability of reliable and accurate methods for serosurveillance studies to understand the disease burden. Therefore, we conducted study to harmonize anti-Zika IgG antibody detection assays with 1st WHO International Standard (16/352) and working standard (16/320) for anti-Zika virus antibody.Additionally, evaluated NuGenTMZIKA-IgG and NovaLisa®ZIKA virus IgG-Capture ELISA using a panel of 278 seraFurther, 106 samples positive for other-flavi viruses were taken for assessing cross-reactivity of the assay, all serums were further tested by Zika-PRNT. The results of this study indicates satisfactory performance of both the assays. Serological and neutralization assays were calibrated according to the international standards. This will help in understanding antibody dynamics in serosurveillance and vaccine studies. However the performance of the kits with possibilities of cross-reactivity will have to be verified by coupling ZIKV and DENV specific ELISA.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.

Hyperendemic Dengue and Possible Zika Circulation in the Westernmost Region of the Indonesian Archipelago.

The transmission of dengue and other medically important mosquito-borne viruses in the westernmost region of Indonesia is not well described. We assessed dengue and Zika virus seroprevalence in Aceh province, the westernmost area of the Indonesian archipelago. Serum samples collected from 199 randomly sampled healthy residents of Aceh Jaya in 2017 were analyzed for neutralizing antibodies by plaque reduction neutralization test (PRNT). Almost all study participants (198/199; 99.5%) presented with multitypic profiles of neutralizing antibodies to two or more DENV serotypes, indicating transmission of multiple DENV in the region prior to 2017. All residents were exposed to one or more DENV serotypes by the age of 30 years. The highest geometric mean titers were measured for DENV-4, followed by DENV-1, DENV-2 and DENV-3. Among a subset of 116 sera, 27 neutralized ZIKV with a high stringency (20 with PRNT90 > 10 and 7 with PRNT90 > 40). This study showed that DENV is hyperendemic in the westernmost region of the Indonesian archipelago and suggested that ZIKV may have circulated prior to 2017.

Zika Virus Infection in the Ovary Induces a Continuously Elevated Progesterone Level and Compromises Conception in Interferon Alpha/Beta Receptor-Deficient Mice.

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impact of ZIKV infection on nonpregnant female reproductive health is not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/ß receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia, and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis, including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months postinfection, damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE Zika virus (ZIKV), a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barré syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on the genital tract of nonpregnant females. This study investigated the effects of ZIKV on the ovaries in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

Serological Evidence of Zika Virus Infection in Febrile Patients and Healthy Blood Donors in Sabah, Malaysian Borneo, 2017-2018.

Several Zika virus (ZIKV) seroprevalence studies have been conducted in Africa, Asia, Oceania, the Americas, and the Caribbean. However, studies on ZIKV seroprevalence are limited in Malaysia though several studies have shown that the disease is endemic in the Malaysian state of Sabah. To evaluate the seroprevalence of ZIKV infection, 818 serum samples were collected from febrile patients and healthy blood donors from the Kudat and Kota Kinabalu districts in Sabah from 2017 to 2018. They were screened for ZIKV infection by IgM and IgG ELISA, and positive ZIKV IgM samples were subjected to a 90% neutralization test for confirmation. Twenty-four (6% [95% CI 4 to 8]) confirmed and two (0.5% [95% CI 0.13 to 1.8]) probable ZIKV infections were detected among 400 febrile illness patients. Of 418 healthy blood donor samples, six (1.4% [95% CI 0.65 to 3]) were determined as confirmed ZIKV infections and six (1.4% [95% CI 0.65 to 3]) indicated probable ZIKV infection. This is the first study on the seroprevalence of ZIKV infections among patients and healthy blood donors in Sabah. Compared with previous studies in Malaysia, this study shows that the incidence of ZIKV infection has increased. It also suggests that a sero-surveillance system is essential to determine the circulation of ZIKV in Sabah, Malaysia.

The Ability of Zika virus Intravenous Immunoglobulin to Protect From or Enhance Zika Virus Disease.

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers.

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Prevalence of arbovirus antibodies in young healthy adult population in Brazil.

BACKGROUND: The emergence and re-emergence of infectious diseases are a cause for worldwide concern. The introduction of Zika and Chikungunya diseases in the Americas has exposed unforeseen medical and logistical challenges for public health systems. Moreover, the lack of preventive measures and vaccination against known and emerging mosquito-transmitted pathogens, and the occurrence of unanticipated clinical complications, has had an enormous social and economic impact on the affected populations. In this study, we aimed to measure the seroprevalence of endemic and emerging viral pathogens in military personnel stationed in Manaus, Amazonas state. METHODS: We measured the seropositivity of antibodies against 19 endemic and emerging viruses in a healthy military personnel group using a hemagglutination inhibition assay (HIA). RESULTS: Overall, DENV positivity was 60.4%, and 30.9% of the individuals reacted against ZIKV. Also, 46.6%, 54.7%, 51.3% and 48.7% individuals reacted against West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV), respectively. Individuals with high DENV HIA titer reacted more frequently with ZIKV or WNV compared to those with low HIA titers. Observed cross-reactivity between Flaviviruses varied depending on the virus serogroup. Additionally, 0.6% and 0.3% individuals were seropositive for Oropouche virus (OROV) and Catu virus (CATUV) from the family Peribunyaviridae, respectively. All samples were negative for Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalomyelitis virus (WEEV), Mayaro virus (MAYV), Mucambo virus (MUCV) and CHIKV from the family Togaviridae. CONCLUSIONS: A high proportion of individuals in our high-risk population (~ 60%) lacked antibodies against major endemic and emerging viruses, which makes them susceptible for further infections. Military personnel serving in the Amazon region could serve as sentinels to strengthen global infectious disease surveillance, particularly in remote areas.

Serological Differences after Acute Zika Virus Infections between Children and Adults: Implication for Use of a Serological Test.

Information is limited regarding differential serological responses after acute Zika virus (ZIKV) infections and prevalence of cross-reactivity with anti-dengue virus (DENV) assays comparing children and adults. Early convalescent sera from a cohort of suspected mild DENV cases between December 2016 and September 2018 at Bamrasnaradura Infectious Diseases Institute in Thailand were tested for nonstructural protein 1 (NS1)-based anti-ZIKV IgM and IgG ELISAs (Euroimmun), and in-house anti-DENV IgM- and IgG-capture ELISAs. ZIKV cases were identified by positive real-time reverse transcriptase-polymerase chain reaction on urine. Sera from 26 (10 children and 16 adults) ZIKV and 227 (153 children and 74 adults) non-ZIKA cases collected at the median duration of 18 days (interquartile range [IQR] 18,19) post-onset of symptoms were tested. Comparing pediatric ZIKV to adult ZIKV cases, the mean anti-ZIKV IgM ratio was higher (2.12 versus 1.27 units, respectively; P = 0.07), whereas mean anti-ZIKV IgG ratio was lower (3.13 versus 4.24 units, respectively; P = 0.03). Sensitivity of anti-ZIKV IgM and specificity of anti-ZIKV IgG in pediatric ZIKV were higher than in adult ZIKV cases (80.0% versus 43.7% and 79.1% versus 43.2%, respectively). No cross-reactivity with anti-DENV IgM- and IgG-capture ELISA were reported in pediatric ZIKV cases in our study, whereas 25% and 12.5% were found in adult ZIKV cases, respectively. Age-related ZIKV serological differences have been observed. Positive NS1-based anti-ZIKV IgM and IgG ELISA at the early convalescent phase could be useful for ZIKV diagnosis in children, even in a dengue endemic setting.

Non-structural protein 1 from Zika virus: Heterologous expression, purification, and potential for diagnosis of Zika infections.

Zika virus (ZIKV) infections were associated with neurological disorders only after the Brazilian outbreak in 2015. The lack of vaccines and precise diagnosis requires a precise method to detect ZIKV infection. This study aimed to evaluate three ZIKV recombinant proteins for the development of ZIKV infections. Here, it was purified stable recombinant ZIKV Capsid (r-ZIKV-c), non-structural proteins NS1 (r-ZIKV-NS1), and NS3 (r-ZIKV-NS3) for detection of the infection by ZIKV in blood sera of patients. A commercial polyclonal antibody recognized the r-ZIKV-NS1. Here, among three proteins, NS1 showed the best result for diagnostic purposes using serum samples, despite the high similarity with NS1 from DENV, and could differentiate the infections. The recombinant NS1 was used to produce a monoclonal antibody to differentiate between DENV and ZIKV NS1. As for recombinant proteins, the result for r-ZIKV-NS1 values showed 77% and 100% sensitivity and specificity, respectively, in the IgM assay. Our data showed the protein could successfully differentiate between sera of ZIKV infected patients from sera of those not infected with the virus and differentiate from sera of DENV infected patients. Thus, the generated recombinant proteins have great potential for serological diagnosis of ZIKV in Brazil, where it is indispensable.

Seroprevalence of Yellow fever, Chikungunya, and Zika virus at a community level in the Gambella Region, South West Ethiopia.

Yellow fever (YF), Chikungunya (CHIK), and Zika(ZIK) are among re-emerging arboviral diseases of major public health concern. Despite the proximity of the Gambella Region to South Sudan where arboviral cases have been recorded repeatedly the current epidemiological situation is unclear in this part of southwest Ethiopia. Therefore, we conducted a community-based seroprevalence survey of YF virus (YFV), CHIK virus (CHIKV), and ZIK virus (ZIKV) infections in two selected districts. A cross-sectional study was conducted in two locations of the Gambella region (Lare and Itang) to investigate the seroprevalence of these viruses' infections. Blood samples were collected from the study participants and screened for IgG antibodies specific to YFV and CHIKV infections using enzyme-linked immunosorbent assays (ELISA). For the detection of ZIKV specific IgG antibodies, Blockade-of-binding ELISA was used. Data were analyzed using the STATA version 13.1 Softwares. A total of 150 individuals (96 males and 54 females, age ranging from 18 to 65 years, mean age ± SD = 35.92 ± 10.99) participated and provided blood samples. Among the 150 samples 135, 90, and 150 were screened for YFV, CHIKV, and ZIKV, respectively. Hence, 2.9% (95% CI: 1.1-7.7%), 15.6% (95% CI: 9.3-24.8%), and 27.3% (95% CI: 20.7-35.3%) of samples tested positive for IgG antibodies to YFV, CHIKV, and ZIKV infections, respectively. Among the individual seropositive for ZIKV, YFV and CHIKV, only six, one and three had a history of residence outside the Gambella region respectively. Agro-pastoral occupation was significantly associated with a higher prevalence of IgG against CHIKV (AOR = 14.17; 95%CI: 2.30, 87.30) and residency in the Lare district (AOR = 11; 95%CI: 3.31, 39.81) was found to be significantly associated with a higher prevalence of IgG against ZIKV. Our findings revealed the occurrence of YFV, CHIKV and ZIKV infections in the study locations.

Zika virus isolation, propagation, and quantification using multiple methods.

Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106-107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10-100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.