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.

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.

BACKGROUND: Japanese encephalitis (JE) virus is recognized as a major cause of encephalitis in Bangladesh. The World Health Organization (WHO) recommends human immunization as the most effective means to control JE. Several WHO-prequalified vaccines are available to prevent JE but no vaccination program has been implemented in Bangladesh. METHODS: We conducted hospital-based surveillance for acute meningitis-encephalitis syndrome (AMES) to describe JE epidemiology and help inform policy decisions about possible immunization strategies for Bangladesh. RESULTS: During 2007-2016, a total of 6,543 AMES patients were identified at four tertiary hospitals. Of the 6,525 patients tested, 548 (8%) were classified as JE cases. These 548 patients resided in 36 (56%) out of 64 districts of Bangladesh, with the highest proportion of JE cases among AMES patients (12% and 7%) presenting at two hospitals in the northwestern part of the country. The median age of JE cases was 30 years, and 193 (35%) were aged ≤15 years. The majority of JE cases (80%) were identified from July through November. CONCLUSIONS: Surveillance results suggest that JE continues to be an important cause of meningo-encephalitis in Bangladesh. Immunization strategies including JE vaccine introduction into the routine childhood immunization program or mass vaccination in certain age groups or geographic areas need to be examined, taking into consideration the cost-effectiveness ratio of the approach and potential for decreasing disease burden.

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.

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.

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.

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.

Chikungunya fever (CHIK) is an acute viral infection caused by infection with chikungunya virus (CHIKV). The disease affects people in areas where certain Aedes species mosquito vectors are present, especially in tropical and subtropical countries. Indonesia has witnessed CHIK disease since the early 1970s with sporadic outbreaks occurring throughout the year. The CHIK clinical manifestation, characterized by fever, headache, and joint pain, is similar to that of dengue (DEN) disease. During a molecular study of a DEN outbreak in Jambi, Sumatra, in the early 2015, DEN-negative samples were evaluated for evidence of CHIKV infection. Among 103 DEN-negative samples, eight samples were confirmed (7.8%) as positive for CHIK by both molecular detection and virus isolation. The mean age of the CHIK patients was 21.3 +/- 9.1 (range 11-35 years). The clinical manifestations of the CHIK patients were mild and mimicked DEN, with fever and headache as the main symptoms. Only three out of eight patients presented with classical joint pain. Sequencing of the envelope glycoprotein E1 gene and phylogenetic analysis identified all CHIKV isolates as belonging to the Asian genotype. Overall, our study confirms sustained endemic CHIKV transmission and the presence of multiple arboviruses circulating during a DEN outbreak in Indonesia. The co-circulation of arboviruses poses a public health threat and is likely to cause misdiagnosis and underreporting of CHIK in DEN-endemic areas such as Indonesia.

Background An outbreak of Guillain-Barre syndrome (GBS), a disorder characterized by acute, symmetric limb weakness with decreased or absent deep-tendon reflexes, was reported in Barranquilla, Colombia, after the introduction of Zika virus in 2015. We reviewed clinical data for GBS cases in Barranquilla and performed a case-control investigation to assess the association of suspect and probable Zika virus disease with GBS. Methods We used the Brighton Collaboration Criteria to confirm reported GBS patients in Barranquilla during October 2015-April 2016. In April 2016, two neighborhood and age range-matched controls were selected for each confirmed GBS case-patient. We obtained demographics and antecedent symptoms in the 2-month period before GBS onset for case-patients and the same period for controls. Sera were collected for Zika virus antibody testing. Suspected Zika virus disease was defined as a history of rash and >= 2 other Zika-related symptoms (fever, arthralgia, myalgia, or conjunctivitis). Probable Zika virus disease was defined as suspected Zika virus disease with laboratory evidence of a recent Zika virus or flavivirus infection. Conditional logistic regression adjusted for sex and race/ethnicity was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Results We confirmed 47 GBS cases. Incidence increased with age (10-fold higher in those >= 60 years versus those < 20 years). We interviewed 40 case-patients and 79 controls. There was no significant difference in laboratory evidence of recent Zika virus or flavivirus infection between case-patients and controls (OR: 2.2; 95% CI: 0.9-5.1). GBS was associated with having suspected (OR: 3.0, 95% CI: 1.1-8.6) or probable Zika virus disease (OR: 4.6, CI: 1.1-19.0). Conclusions Older individuals and those with suspected and probable Zika virus disease had higher odds of developing GBS. Key points We confirmed a Guillain-Barre syndrome (GBS) outbreak in Barranquilla, Colombia, during October 2015-April 2016. A case-control investigation using neighborhood controls showed an association of suspected and probable Zika virus disease with GBS.

Understanding the ability of the chikungunya virus (CHIKV) to be transmitted by Aedes vectors in the Americas is critical for assessing epidemiological risk. One element that must be considered is the minimum infectious dose of virus that can lead to transmission following the extrinsic incubation period. This study aimed to determine the minimum infection rate for the two Aedes species studied. The results revealed that doses as low as 3.9 log10 plaque-forming units per mL (pfu/mL) of an Asian genotype CHIKV strain can lead to transmission by Ae. albopictus, and doses of at least 5.3 log10 pfu/mL from the same strain are needed for transmission from Ae. aegypti. These low infecting doses suggest that infected individuals may be infectious for almost the entire period of their viremia, and therefore, to prevent further cases, measures should be taken to prevent them from getting bitten by mosquitoes during this period.

In mid-2015, Salvador, Brazil, reported an outbreak of Guillain-Barre syndrome (GBS), coinciding with the introduction and spread of Zika virus (ZIKV). We found that GBS incidence during April-July 2015 among those ≥12 years of age was 5.6 cases/100,000 population/year and increased markedly with increasing age to 14.7 among those ≥60 years of age. We conducted interviews with 41 case-patients and 85 neighborhood controls and found no differences in demographics or exposures prior to GBS-symptom onset. A higher proportion of case-patients (83%) compared to controls (21%) reported an antecedent illness (OR 18.1, CI 6.9-47.5), most commonly characterized by rash, headache, fever, and myalgias, within a median of 8 days prior to GBS onset. Our investigation confirmed an outbreak of GBS, particularly in older adults, that was strongly associated with Zika-like illness and geo-temporally associated with ZIKV transmission, suggesting that ZIKV may result in severe neurologic complications.

Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies.

Highlands J virus (HJV) is an alphavirus closely related to western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV). HJV is an avian pathogen with the potential for disruption of poultry operations, but is not known to cause human or equine disease. HJV has only been identified in the eastern United States and is thought to have a transmission cycle similar to that of EEEV involving Culiseta melanura mosquitoes and birds. However, HJV is more genetically similar to WEEV and it remains unclear if it may be transmitted by Culex species mosquitoes like WEEV. Seven strains of HJV were characterized to assess this potential. Phylogenetic analysis of whole genome sequences revealed four distinct HJV lineages (lineages 1-4), and vector competence studies in Cx. tarsalis with four of the HJV strains from different lineages yielded two distinct infection patterns. Lineage 1 strains had low infection rates, while lineages 2 and 4 had significantly higher infection rates similar to those previously published for WEEV. The average mosquito body viral titer was highest at 8 dpi (6.60-7.26 log10 pfu equivalents/body), and head titers at all time points ranged between 6.01 and 6.80 log10 pfu equivalents/head. Nearly 45% of mosquitoes infected with strain AB-80-9 were able to transmit virus in saliva with an average titer of 5.02 log10 pfu equivalents/saliva. A single amino acid difference between high and low infectivity phenotypes was identified at genome position 8605, in the E2 gene. A nonpolar glycine was present in the low infectivity lineage 1 strains, while an acidic glutamic acid was present in the higher infectivity lineage 2 and 4 strains. This study demonstrates HJV transmission by Cx. tarsalis mosquitoes and clearly identifies the potential for transmission in the western United States. Two infection phenotypes were exhibited, indicating the need for further studies to understand Culex species transmission patterns.

Having a mechanism to assess the transmission dynamics of a vector-borne virus is one critical component of understanding the life cycle of these viruses. Laboratory infection systems using artificial blood meals is one valuable approach for monitoring the progress of virus in its mosquito host and evaluating potential points for interruption of the cycle for control purposes. Here, we describe an artificial blood meal system with Chikungunya virus (CHIKV) and the processing of mosquito tissues and saliva to understand the movement and time course of virus infection in the invertebrate host.

Arthropodborne viruses (arboviruses) cause substantial human disease worldwide and have a pronounced effect on public health throughout Asia. Zika virus, discovered in Uganda in 1947 (1), is a flavivirus related to the following viruses: dengue (DENV), West Nile, Japanese encephalitis, and yellow fever. Like DENV, Zika virus is transmitted by Aedes mosquitoes. Zika virus emerged as a public health problem in 2007, when it caused an epidemic in Micronesia (2). Since then, the virus has caused epidemics elsewhere in the Pacific islands (3) and recently emerged in South America (4). Zika virus has been reported to cause mild and self-limited infection that can be misdiagnosed as dengue because of similar clinical features and serologic cross-reactivity (2). Zika virus has not, however, been reported to cause substantial thrombocytopenia or result in the serious vascular leakage that can be fatal in DENV infection. | Until recently, most evidence for Zika virus infection in Asia, including in Indonesia (5), has been serologic, but recent virus strains isolated from persons in Thailand (6), the Philippines (7), and Cambodia (8) have begun to clarify its genomic diversity. Phylogenetically, Zika virus appears to have 2 major lineages, African and Asian (9).

Zika virus is an emerging mosquito-borne flavivirus. Recent outbreaks of Zika virus disease in the Pacific Islands and the Region of the Americas have identified new modes of transmission and clinical manifestations, including adverse pregnancy outcomes. However, data on the epidemiology and clinical findings of laboratory-confirmed Zika virus disease remain limited. During January 1, 2015-February 26, 2016, a total of 116 residents of 33 U.S. states and the District of Columbia had laboratory evidence of recent Zika virus infection based on testing performed at CDC. Cases include one congenital infection and 115 persons who reported recent travel to areas with active Zika virus transmission (n = 110) or sexual contact with such a traveler (n = 5). All 115 patients had clinical illness, with the most common signs and symptoms being rash (98%; n = 113), fever (82%; 94), and arthralgia (66%; 76). Health care providers should educate patients, particularly pregnant women, about the risks for, and measures to prevent, infection with Zika virus and other mosquito-borne viruses. Zika virus disease should be considered in patients with acute onset of fever, rash, arthralgia, or conjunctivitis, who traveled to areas with ongoing Zika virus transmission (http://www.cdc.gov/zika/geo/index.html) or who had unprotected sex with a person who traveled to one of those areas and developed compatible symptoms within 2 weeks of returning.

BACKGROUND: Data regarding the viremia profile of chikungunya virus (CHIKV) infected patients especially during the pre-febrile period is limited. OBJECTIVE: To obtain virological kinetic data on CHIKV infections. STUDY DESIGN: A two-week community observation for dengue transmission was conducted in Bandung, Indonesia, from 2005 to 2009. Acute specimens from non-dengue febrile patients were screened by pan-alphavirus conventional RT-PCR. The positives were confirmed for CHIKV RNA by a specific RT-PCR followed by sequencing. Simultaneously these specimens were also cultured in Vero cells and tested for anti-CHIK IgM MAC-ELISA. All the available serial specimens,including the pre-febrile specimens, from confirmed CHIK cases, were tested by virus isolation, RT-PCR, qRT-PCR, and CHIK IgM ELISA. RESULTS: There were five laboratory confirmed CHIK cases identified and studied. Among these, viremia was determined to extend from as early as 6 days prior to until 13 days post fever onset. Quantitative RT-PCR showed viremia peaked at or near onset of illness. CONCLUSION: In this study, individuals were identified with viremia prior to fever onset and extending beyond the febrile phase. This extended viremic phase has the potential to impact transmission dynamics and thus the public health response to CHIK outbreaks.

Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses.

The first known outbreak of eastern equine encephalitis (EEE) in Vermont occurred on an emu farm in Rutland County in 2011. The first isolation of EEE virus (EEEV) in Vermont (VT11) was during this outbreak. Phylogenetic analysis revealed that VT11 was most closely related to FL01, a strain from Florida isolated in 2001, which is both geographically and temporally distinct from VT11. EEEV RNA was not detected in any of the 3,905 mosquito specimens tested, and the specific vectors associated with this outbreak are undetermined.

Two species of Aedes (Stegomyia) were collected in response to the first chikungunya virus (CHIKV) outbreak on Yap Island: the native species Ae. hensilli Farner and the introduced species Ae. aegypti (L.). Fourteen CHIKV-positive mosquito pools were detected. Six pools were composed of female Ae. hensilli, six pools were composed of female Ae. aegypti, one pool was composed of male Ae. hensilli, and one pool contained female specimens identified as Ae. (Stg.) spp. Infection rates were not significantly different between female Ae. hensilli and Ae. aegypti. The occurrence of human cases in all areas of Yap Island and the greater number of sites that yielded virus from Ae. hensilli combined with the ubiquitous distribution of this species incriminate Ae. hensilli as the most important vector of CHIKV during the outbreak. Phylogenic analysis shows that virus strains on Yap are members of the Asia lineage and closely related to strains currently circulating in the Caribbean.

An epidemic of Zika virus (ZIKV) illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s) involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s). Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at select sites around the capital city. The predominant species found on the island was Aedes (Stegomyia) hensilli. No virus isolates were obtained from the adult field material collected, nor did any of the immature mosquitoes that were allowed to emerge to adulthood contain viable virus or nucleic acid. Therefore, laboratory studies of the probable vector, Ae. hensilli, were undertaken to determine the likelihood of this species serving as a vector for Zika virus and other arboviruses. Infection rates of up to 86%, 62%, and 20% and dissemination rates of 23%, 80%, and 17% for Zika, chikungunya, and dengue-2 viruses respectively, were found supporting the possibility that this species served as a vector during the Zika outbreak and that it could play a role in transmitting other medically important arboviruses.

Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11,056 kb in length and contains the five core rhabdovirus genes plus an additional C gene (within the open reading frame of the phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells resulted in significant growth, with a peak titer of 7.8 log10 PFU/mL observed in baby hamster kidney cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes demonstrating a 47.4% infection rate in the body but no dissemination to the salivary glands; this suggests that this novel virus is not arthropod-borne like some other members of the family Rhabdoviridae.

We report the presence of West Nile virus in a cryopreserved, dengue-negative serum specimen collected from an acute fever case on Java in 2004-2005. The strain belongs to genotype lineage 2, which has recently been implicated in human outbreaks in Europe.

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high ( approximately 90-100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.