Background: Usutu virus (USUV) is an emerging flavivirus, closely related to West Nile virus (WNV), that has spread into Europe from Africa. Since Culex tarsalis Coquillett is an important vector for WNV transmission in the United States, we tested the ability of USUV to replicate in and be transmitted by these mosquitoes. Materials and Methods: USUV was used to infect 3-4 day-old Cx. tarsalis with 5.6 to 7.5 log(10) pfu/ml in goose bloodmeals. Saliva, heads, and bodies were collected on day 13 or 14 and analyzed by RT-qPCR for detection for USUV vRNA. Blotting paper punches were also collected daily to assess viral transmissibility. Results: The low and high dose blood meal resulted in 0% and 19.6% of the mosquitoes having established infections, respectively. All of the high dose had a dissemination of USUV RNA to the heads and none of the filter papers had detectable USUV RNA, but five of the capillary saliva collections were positive, representing 45.5% of the infected mosquitoes. Conclusions: Limited infection of Cx. tarsalis was observed when exposed to bloodmeals with greater than 107 pfu/mL of USUV, indicating this vector is not likely to have a key role in transmission of the virus.

Central nervous system (CNS) viral infections are critical causes of morbidity and mortality in children; however, comprehensive data on etiology is lacking in developing countries such as Indonesia. To study the etiology of CNS infections in a pediatric population, 50 children admitted to two hospitals in Bandung, West Java, during 2017-2018 were enrolled in a CNS infection study. Cerebrospinal fluid and serum specimens were tested using molecular, serological, and virus isolation platforms for a number of viral and bacteriological agents. Causal pathogens were identified in 10 out of 50 (20%) and included cytomegalovirus (n = 4), Streptococcus pneumoniae (n = 2), tuberculosis (n = 2), Salmonella serotype Typhi (n = 1) and dengue virus (n = 1). Our study highlights the importance of using a wide range of molecular and serological detection methods to identify CNS pathogens, as well as the challenges of establishing the etiology of CNS infections in pediatric populations of countries with limited laboratory capacity.

Background: Assessing the potential for mosquitoes to transmit medically important arboviruses is essential for understanding their threat to human populations. Currently, vector competence studies are typically performed by collecting saliva using a glass capillary tube system which involves sacrificing the mosquito at the time of saliva collection allowing only a single data point. These techniques also require handling infected mosquitoes and glass capillaries, constituting a safety risk. Materials and Methods: To improve the efficiency and safety of assessing vector competence, a novel containment and saliva collection approach for individually housed mosquitoes was developed. The improved housing, allowing longitudinal tracking of individual mosquitoes, consists of a 12-well Corning polystyrene plate sealed with a three-dimensional printed lid that holds organdy netting firmly against the rims of the wells. Results: This method provides excellent mosquito survival for five species of mosquitoes, with at least 79% of each species tested surviving for more than 2 weeks, comparable to the carton survival rates of ≥76%. When the plate housing system was used to assess vector infection, replication of West Nile virus (WNV) in mosquito tissues was similar to traditional containment mosquito housing. Mosquito saliva was collected using either blotting paper pads or traditional glass capillaries to assay viral transmission. The blotting paper collection showed similar or better sensitivity than the capillary method; in addition, longitudinal saliva samples could be collected from individual mosquitoes housed in the 12-well plates. Conclusions: The improved housing and saliva collection technique described herein provides a safer and more informative method for determining vector competence in mosquitoes.

The Togaviridae family, genus, Alphavirus, includes several mosquito-borne human pathogens with the potential to spread to near pandemic proportions. Most of these are zoonotic, with spillover infections of humans and domestic animals, but a few such as chikungunya virus (CHIKV) have the ability to use humans as amplification hosts for transmission in urban settings and explosive outbreaks. Most alphaviruses cause nonspecific acute febrile illness, with pathogenesis sometimes leading to either encephalitis or arthralgic manifestations with severe and chronic morbidity and occasional mortality. The development of countermeasures, especially against CHIKV and Venezuelan equine encephalitis virus that are major threats, has included vaccines and antibody-based therapeutics that are likely to also be successful for rapid responses with other members of the family. However, further work with these prototypes and other alphavirus pathogens should target better understanding of human tropism and pathogenesis, more comprehensive identification of cellular receptors and entry, and better understanding of structural mechanisms of neutralization.

The first three SARS-CoV-2 phylogenetic lineages classified as variants of concern (VOCs) in the United States (U.S.) from December 15, 2020 to February 28, 2021, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1) lineages, were initially detected internationally. This investigation examined available travel history of coronavirus disease 2019 (COVID-19) cases reported in the U.S. in whom laboratory testing showed one of these initial VOCs. Travel history, demographics, and health outcomes for a convenience sample of persons infected with a SARS-CoV-2 VOC from December 15, 2020 through February 28, 2021 were provided by 35 state and city health departments, and proportion reporting travel was calculated. Of 1,761 confirmed VOC cases analyzed, 1,368 had available data on travel history. Of those with data on travel history, 1,168 (85%) reported no travel preceding laboratory confirmation of SARS-CoV-2 and only 105 (8%) reported international travel during the 30 days preceding a positive SARS-CoV-2 test or symptom onset. International travel was reported by 92/1,304 (7%) of persons infected with the Alpha variant, 7/55 (22%) with Beta, and 5/9 (56%) with Gamma. Of the first three SARS-CoV-2 lineages designated as VOCs in the U.S., international travel was common only among the few Gamma cases. Most persons infected with Alpha and Beta variant reported no travel history, therefore, community transmission of these VOCs was likely common in the U.S. by March 2021. These findings underscore the importance of global surveillance using whole genome sequencing to detect and inform mitigation strategies for emerging SARS-CoV-2 VOCs.

As a part of a systematic study of mosquitoes and associated viruses in Uganda, a virus was isolated from a pool of Mansonia uniformis collected in July 2017, in the Kitgum District of northern Uganda. Sequence analysis determined that the virus is Yata virus (YATAV; Ephemerovirus yata; family Rhabdoviridae). The only previous reported isolation of YATAV was in 1969 in Birao, Central African Republic, also from Ma. uniformis mosquitoes. The current sequence is over 99% identical at the nucleotide level to the original isolate, indicating a high level of YATAV genomic stability.

Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.

The reservoir for zoonotic o'nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats.

Despite causing numerous large outbreaks in the 20th century, few isolates of o'nyong nyong virus (ONNV) have been fully sequenced. Here, we report the complete genome sequence of an isolate of ONNV obtained from a febrile patient in northwest Uganda in 2017, designated ONNV UVRI0804.

Chikungunya virus (CHIKV) is recognized but rarely considered as a cause of central nervous system infection in endemic areas. A total of 244 patients with acute meningoencephalitis in Indonesia were retrospectively tested to identify whether any CHIKV infection was associated with neurological manifestations, especially in provinces known for CHIKV endemicity. Cerebrospinal fluid and blood specimens were tested using CHIKV-specific real-time reverse transcription polymerase chain reaction and IgM ELISA, alongside a panel of neurotropic viruses. We report four cases of suspected or confirmed CHIKV-associated neurological disease, including CHIKV RNA detection in cerebrospinal fluid (CSF) of one patient and in acute serum of another, and CHIKV IgM in CSF of three patients and in serum of a fourth. In conclusion, CHIKV should be considered as a cause of neurologic disease in endemic areas and especially during outbreaks, in addition to the more common arboviral diseases such as dengue and Japanese encephalitis viruses.

Eastern equine encephalitis virus (EEEV; Family Togaviridae), is an endemic pathogen first isolated in 1933 with distribution primarily in the eastern US and Canada. The virus has caused periodic outbreaks in both humans and equines along the eastern seaboard and through the southern coastal states. While the outbreaks caused by EEEV have been sporadic and varied geographically since the discovery of the virus, it has continued to expand its range moving into the Midwest states as well. Additionally, one of the largest outbreaks was recorded in 2019 prompting concerns that outbreaks were becoming larger and more frequent. Because the virus can cause serious disease and because it is transmissible by both mosquitoes and aerosol, there has been renewed interest in identifying potential options for vaccines. Currently, there are no licensed vaccines and control relies completely on the use of personal protective measures and integrated vector control which have limited effectiveness for the EEEV vectors. Several vaccine candidates are currently being developed; this review will describe the multiple options under consideration for future development and assess their relative advantages and disadvantages.

Chikungunya fever is an acute febrile illness that is often associated with severe polyarthralgia in humans. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus. Since its reemergence in 2004, the virus has spread throughout the tropical world and several subtropical areas affecting millions of people to become a global public health issue. Given the significant disease burden, there is a need for medical countermeasures and several vaccine candidates are in clinical development. To characterize the global epidemiology of chikungunya and inform vaccine development, we undertook a systematic literature review in MEDLINE and additional public domain sources published up to June 13, 2020 and assessed epidemiological trends from 1999 to 2020. Observational studies addressing CHIKV epidemiology were included and studies not reporting primary data were excluded. Only descriptive analyses were conducted. Of 3,883 relevant sources identified, 371 were eligible for inclusion. 46% of the included studies were published after 2016. Ninety-seven outbreak reports from 45 countries and 50 seroprevalence studies from 31 countries were retrieved, including from Africa, Asia, Oceania, the Americas, and Europe. Several countries reported multiple outbreaks, but these were sporadic and unpredictable. Substantial gaps in epidemiological knowledge were identified, specifically granular data on disease incidence and age-specific infection rates. The retrieved studies revealed a diversity of methodologies and study designs, reflecting a lack of standardized procedures used to characterize this disease. Nevertheless, available epidemiological data emphasized the challenges to conduct vaccine efficacy trials due to disease unpredictability. A better understanding of chikungunya disease dynamics with appropriate granularity and better insights into the duration of long-term population immunity is critical to assist in the planning and success of vaccine development efforts pre and post licensure.

Central nervous system involvement of dengue virus is increasingly reported from endemic areas. This study describes the clinical characteristics and laboratory features of a pediatric patient enrolled in a central nervous system illness study conducted in 2017–2018 to identify viral and bacterial etiologies in Indonesian children. Dengue diagnostics including molecular and serological testing were performed on an encephalitis patient who presented with both classical dengue and neurological clinical symptoms. Dengue virus serotype 1 RNA was detected in both cerebrospinal fluid and serum by serotype-specific reverse transcription polymerase chain reaction, and the E gene was successfully sequenced. Anti-dengue virus immunoglobulin M was detected in both admission and discharge sera, whereas anti-dengue virus immunoglobulin G was identified only in the discharge serum. This study describes the central nervous system complications in a case with dengue virus infection in West Java, Indonesia, and highlights the potential for dengue virus serotype 1, a serotype rarely associated with neurotropism, to cause encephalitis. © The Author(s) 2021.

We would like to respond to the letter by Zhang and others regarding our study published in the American Journal of Tropical Medicine and Hygiene.1 Our serum samples were all stored in annually calibrated –80°C freezers. The samples were only thawed once. In general, antibodies are known to remain stable in frozen storage over lengthy periods. There are numerous publications regarding antibody stability during storage,2–6 and we believe that all antibodies, including antibodies against Zika virus, will remain stable during storage.

The presence of Zika virus (ZIKV) in Indonesia has been recognized since the 1970s, but its transmission dynamics there have been poorly understood. To understand more fully the geographic distribution and burden of ZIKV disease, we performed retrospective serological tests on specimens collected from asymptomatic children age 5 to 9 years old living at 30 sites in 14 provinces. Of 870 serum samples tested, 9.2% were found to be positive for anti-ZIKV antibodies, as confirmed by plaque reduction neutralization assays. This was the same overall prevalence reported previously for 1- to 4-year-old children collected at the same sites at the same time. Together with geographic differences in seroprevalence between the age groups, these data suggest that, although ZIKV might be endemic in Indonesia, its occurrence has been focal and episodic.

BACKGROUND: Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. METHODOLOGY/PRINCIPAL FINDINGS: Four hundred and seventy nine acutely febrile patients presenting between September 2017-2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4-3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. CONCLUSIONS/SIGNIFICANCE: Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia.

Although Japanese encephalitis virus (JEV) is considered endemic in Indonesia, there are only limited reports of JEV infection from a small number of geographic areas within the country with the majority of these being neuroinvasive disease cases. Here, we report cases of JEV infection in non-encephalitic acute febrile illness patients from Bali, Indonesia. Paired admission (S1) and discharge (S2) serum specimens from 144 acute febrile illness patients (without evidence of acute dengue virus infection) were retrospectively tested for anti-JEV IgM antibody and confirmed by plaque reduction neutralization test (PRNT) for JEV infection. Twenty-six (18.1%) patients were anti-JEV IgM-positive or equivocal in their S2 specimens, of which 5 (3.5%) and 8 (5.6%) patients met the criteria for confirmed and probable JEV infection, respectively, based on PRNT results. Notably, these non-encephalitic JE cases were less likely to have thrombocytopenia, leukopenia, and lower hematocrit compared with confirmed dengue cases of the same cohort. These findings highlight the need to consider JEV in the diagnostic algorithm for acute febrile illnesses in endemic areas and suggest that JEV as a cause of non-encephalitic disease has likely been underestimated in Indonesia.

BACKGROUND: Rickettsia felis has recently emerged worldwide as a cause of human illness. Typically causing mild, undifferentiated fever, it has been implicated in several cases of non-fatal neurological disease in Mexico and Sweden. Its distribution and pathogenicity in Southeast Asia is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We retroactively tested cerebrospinal fluid (CSF) or sera from 64 adult patients admitted to hospital in North Sulawesi, Indonesia with acute neurological disease. Rickettsia felis DNA was identified in the CSF of two fatal cases of meningoencephalitis using multi-locus sequence typing semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies to the R. felis reference strain URRWXCal2 for the 17-kDa and ompB genes, and 99.91% to gltA. CONCLUSION/SIGNIFICANCE: The identification of R. felis in the CSF of two fatal cases of meningoencephalitis in Indonesia suggests the distribution and pathogenicity of this emerging vector-borne bacteria might be greater than generally recognized. Typically Rickettsia are susceptible to the tetracyclines and greater knowledge of R. felis endemicity in Indonesia should lead to better management of some acute neurological cases.

Zika virus (ZIKV) has recently been confirmed as endemic in Indonesia, but no congenital anomalies (CA) related to ZIKV infection have been reported. We performed molecular and serological testing for ZIKV and other flaviviruses on cord serum and urine samples collected in October 2016 to April 2017 during a prospective, cross-sectional study of neonates in Jakarta, Indonesia. Of a total of 429 neonates, 53 had CA, including 14 with microcephaly. These 53, and 113 neonate controls without evidence of CA, were tested by ZIKV-specific real-time reverse transcription polymerase chain reaction (RT-PCR), pan-flavivirus RT-PCR, anti-ZIKV and anti-DENV IgM ELISA, and plaque reduction neutralization test. There was no evidence of ZIKV infection among neonates in either the CA or non-CA cohorts, except in three cases with low titers of anti-ZIKV neutralizing antibodies. Further routine evaluation throughout Indonesia of pregnant women and their newborns for exposure to ZIKV should be a high priority for determining risk.

Central nervous system (CNS) viral infections are important causes of morbidity and mortality worldwide but the systematic survey of patients admitted to hospitals with CNS infections in many countries, including Indonesia, is limited. To obtain more information regarding the causes of CNS infections in Indonesia, this study was performed to detect and identify viral agents associated with CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia. Adult patients admitted to R.D. Kandou General Hospital with presumed CNS infection were enrolled. Cerebrospinal fluid, serum, and throat swab samples were collected and tested using molecular, serological, and virus isolation assays. A confirmed viral etiology was established in three and a probable/possible in 11 out of 74 patients. The most common was herpes simplex virus 1 (7/74, 9.5%), followed by Epstein-Barr virus (2/74, 2.7%), cytomegalovirus (1/74, 1.4%), enterovirus D68 (1/74, 1.4%), rhinovirus A (1/74, 1.4%), dengue virus (1/64, 1.6%), and Japanese encephalitis virus (1/64, 1.6%). There were 20 fatal cases (27.0%) during hospitalization in which eight were associated with viral causes. We identified herpes simplex virus 1 as the most common cause of CNS infection among adults in North Sulawesi with most of the cases remaining undiagnosed. Our study highlights the challenges in establishing the etiology of viral CNS infections and the importance of using a wide range of molecular and serological detection methods to identify CNS viruses.

Arthropod-borne viruses (arboviruses) are some of the most important human pathogens in the world yet developing countermeasures to them is challenging and rarely leads to licensed products. A small number of arbovirus vaccines, including yellow fever and Japanese encephalitis vaccines, have long been used to prevent disease.1, 2 In 2016, a dengue vaccine was licensed to aid the control of dengue virus infections, which are responsible for an estimated 2 million severe disease cases and 21 000 deaths each year.3 Another virus transmitted by Aedes aegypti, chikungunya virus, has recently emerged as a global pathogen, with about 50% of the world's population at risk of infection.4 Although chikungunya virus infection is not associated with a high mortality rate, the severe and chronic clinical manifestations associated with the infection have led to the desire for rapid development of vaccines against the virus.5, 6, 7

We assessed Zika virus seroprevalence among healthy 1-4-year-old children using a serum sample collection assembled in 2014 representing 30 urban sites across Indonesia. Of 662 samples, 9.1% were Zika virus seropositive, suggesting widespread recent Zika virus transmission and immunity. Larger studies are needed to better determine endemicity in Indonesia.

The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

Madariaga virus (MADV), an alphavirus in the eastern equine encephalitis antigenic complex, has only rarely been shown to cause human disease even with the known capacity to cause outbreaks in equine populations in Latin America. When human cases have been detected, until recently, most have been associated with severe neurological disease. However, a follow up study to an outbreak of MADV in Panama in 2010 revealed both mild cases, as well as evidence of asymptomatic infections [1]. Additionally, seroprevalence studies in the Amazonia region of Peru showed MADV antibodies in up to 3% of the residents [2], suggesting that infection with this virus may not be uncommon even in the absence of reported disease.

Introduction: A number of arboviruses have previously been isolated from naturally-infected East African bats, however the role of bats in arbovirus maintenance is poorly understood. The aim of this study was to investigate the exposure history of Ugandan bats to a panel of arboviruses. Materials and methods: Insectivorous and fruit bats were captured from multiple locations throughout Uganda during 2009 and 2011-2013. All serum samples were tested for neutralizing antibodies against West Nile virus (WNV), yellow fever virus (YFV), dengue 2 virus (DENV-2), Zika virus (ZIKV), Babanki virus (BBKV), and Rift Valley fever virus (RVFV) by plaque reduction neutralization test (PRNT). Sera from up to 626 bats were screened for antibodies against each virus. Results and Discussion: Key findings include the presence of neutralizing antibodies against RVFV in 5/52 (9.6%) of little epauletted fruit bats (Epomophorus labiatus) captured from Kawuku and 3/54 (5.6%) Egyptian rousette bats from Kasokero cave. Antibodies reactive to flaviviruses were widespread across bat taxa and sampling locations. Conclusion: The data presented demonstrate the widespread exposure of bats in Uganda to arboviruses, and highlight particular virus-bat associations that warrant further investigation.

Background: In 2015, the number of infants born with microcephaly increased in Paraiba, Brazil, after a suspected Zika virus outbreak. We did a retrospective case-control investigation to assess the association of microcephaly and Zika virus. Methods: We enrolled cases reported to the national database for microcephaly and born between Aug 1, 2015, and Feb 1, 2016, on the basis of their birth head circumference and total body length. We identified controls from the national birth registry and matched them to cases by location, aiming to enrol a minimum of two controls per case. Mothers of both cases and controls were asked about demographics, exposures, and illnesses and infants were measured at a follow-up visit 1-7 months after birth. We took blood samples from mothers and infants and classified those containing Zika virus IgM and neutralising antibodies as evidence of recent infection. We calculated prevalence of microcephaly and odds ratios (ORs) using a conditional logistic regression model with maximum penalised conditional likelihood, and combined these ORs with exposure probability estimates to determine the attributable risk. Findings: We enrolled 164 of 706 infants with complete information reported with microcephaly at birth, of whom we classified 91 (55%) as having microcephaly on the basis of their birth measurements, 36 (22%) as small, 21 (13%) as disproportionate, and 16 (10%) as not having microcephaly. 43 (26%) of the 164 infants had microcephaly at follow-up for an estimated prevalence of 5.9 per 1000 livebirths. We enrolled 114 control infants matched to the 43 infants classified as having microcephaly at follow-up. Infants with microcephaly at follow-up were more likely than control infants to be younger (OR 0.5, 95% CI 0.4-0.7), have recent Zika virus infection (21.9, 7.0-109.3), or a mother with Zika-like symptoms in the first trimester (6.2, 2.8-15.4). Once Zika virus infection and infant age were controlled for, we found no significant association between microcephaly and maternal demographics, medications, toxins, or other infections. Based on the presence of Zika virus antibodies in infants, we concluded that 35-87% of microcephaly occurring during the time of our investigation in northeast Brazil was attributable to Zika virus. We estimate 2-5 infants per 1000 livebirths in Paraiba had microcephaly attributable to Zika virus. Interpretation: Time of exposure to Zika virus and evidence of infection in the infants were the only risk factors associated with microcephaly. This investigation has improved understanding of the outbreak of microcephaly in northeast Brazil and highlights the need to obtain multiple measurements after birth to establish if an infant has microcephaly and the need for further research to optimise testing criteria for congenital Zika virus infection. Funding: Centers for Disease Control and Prevention.

Despite evidence of arbovirus activity in northwestern Uganda (West Nile Sub-region), there is very limited information on the mosquito fauna of this region. The only published study reported 52 mosquito species in northwestern Uganda but this study took place in 1950 and the information has not been updated for more than 60 yr. In January and June 2011, CO2 baited-light traps were used to collect 49,231 mosquitoes from four different locations, Paraa (9,487), Chobe (20,025), Sunguru (759), and Rhino Camp (18,960). Overall, 72 mosquito species representing 11 genera were collected. The largest number of distinct species was collected at Chobe (43 species), followed by Paraa (40), Sunguru (34), and Rhino Camp (25). Only eight of the 72 species (11.1%) were collected from all four sites: Aedes (Stegomyia) aegypti formosus (Walker), Anopheles (Cellia) funestus group, Culex (Culex) decens group, Cx. (Culex) neavei Theobald, Cx. (Culex) univittatus Theobald, Cx. (Culiciomyia) cinereus Theobald, Cx. (Oculeomyia) poicilipes (Theobald), and Mansonia (Mansonoides) uniformis (Theobald). Fifty-four species were detected in northwestern Uganda for the first time; however, these species have been detected elsewhere in Uganda and do not represent new introductions to the country. Thirty-three species collected during this study have previously been implicated in the transmission of arboviruses of public health importance.

Although neurological manifestations associated with dengue viruses (DENV) infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3) from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus.

Beginning in 2004, chikungunya virus (CHIKV) went from an endemic pathogen limited to Africa and Asia that caused periodic outbreaks to a global pathogen. Given that outbreaks caused by CHIKV have continued and expanded, serious consideration must be given to identifying potential options for vaccines and therapeutics. Currently, there are no licensed products in this realm, and control relies completely on the use of personal protective measures and integrated vector control, which are only minimally effective. Therefore, it is prudent to urgently examine further possibilities for control. Vaccines have been shown to be highly effective against vector-borne diseases. However, as CHIKV is known to rapidly spread and generate high attack rates, therapeutics would also be highly valuable. Several candidates are currently being developed; this review describes the multiple options under consideration for future development and assesses their relative advantages and disadvantages.

Background. Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive IgM results in areas with varying past dengue virus (DENV) infection incidence. Methods. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from January 1 through August 31, 2016 with positive ZIKAV IgM results and ZIKAV and DENV PRNT performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false-positive for samples interpreted as DENV infection or negative. Results. In US states, 208 (27%) of 759 IgM positives were confirmed as ZIKAV, compared to 11 (21%) of 52 in the US Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM positives were unspecified flavivirus infections. The false-positivity rate was 27% in US states, 18% in USVI, 2% in American Samoa, and 6% in Puerto Rico. Conclusions. In US states, PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM positive samples. Almost a third of ZIKAV IgM positive results did not confirm; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher DENV transmission, PRNT usually could not differentiate between ZIKAV and DENV infections.