Aaron C. Brault

A single positively selected West Nile viral mutation confers increased virogenesis in American crows

West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well-described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.

Evolutionary Relationships and Systematics of the Alphaviruses

ABSTRACT Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus Alphavirus. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.

West Nile Virus: Review of the Literature

IMPORTANCE Since its introduction in North America in 1999, West Nile virus has produced the 3 largest arboviral neuroinvasive disease outbreaks ever recorded in the United States. OBJECTIVE To review the ecology, virology, epidemiology, clinical characteristics, diagnosis, prevention, and control of West Nile virus, with an emphasis on North America. EVIDENCE REVIEW PubMed electronic database was searched through February 5, 2013. United States national surveillance data were gathered from the Centers for Disease Control and Prevention. FINDINGS West Nile virus is now endemic throughout the contiguous United States, with 16,196 human neuroinvasive disease cases and 1549 deaths reported since 1999. More than 780,000 illnesses have likely occurred. To date, incidence is highest in the Midwest from mid-July to early September. West Nile fever develops in approximately 25% of those infected, varies greatly in clinical severity, and symptoms may be prolonged. Neuroinvasive disease (meningitis, encephalitis, acute flaccid paralysis) develops in less than 1% but carries a fatality rate of approximately 10%. Encephalitis has a highly variable clinical course but often is associated with considerable long-term morbidity. Approximately two-thirds of those with paralysis remain with significant weakness in affected limbs. Diagnosis usually rests on detection of IgM antibody in serum or cerebrospinal fluid. Treatment is supportive; no licensed human vaccine exists. Prevention uses an integrated pest management approach, which focuses on surveillance, elimination of mosquito breeding sites, and larval and adult mosquito management using pesticides to keep mosquito populations low. During outbreaks or impending outbreaks, emphasis shifts to aggressive adult mosquito control to reduce the abundance of infected, biting mosquitoes. Pesticide exposure and adverse human health events following adult mosquito control operations for West Nile virus appear negligible. CONCLUSIONS AND RELEVANCE In North America, West Nile virus has and will remain a formidable clinical and public health problem for years to come.

Genome-scale phylogenetic analyses of chikungunya virus reveal independent emergences of recent epidemics and various evolutionary rates.

ABSTRACT Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.

Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors — Final Report

BACKGROUND Ebola virus has been detected in the semen of men after their recovery from Ebola virus disease (EVD). We report the presence of Ebola virus RNA in semen in a cohort of survivors of EVD in Sierra Leone. METHODS We enrolled a convenience sample of 220 adult male survivors of EVD in Sierra Leone, at various times after discharge from an Ebola treatment unit (ETU), in two phases (100 participants were in phase 1, and 120 in phase 2). Semen specimens obtained at baseline were tested by means of a quantitative reverse‐transcriptase–polymerase‐chain‐reaction (RT‐PCR) assay with the use of the target sequences of NP and VP40 (in phase 1) or NP and GP (in phase 2). This study did not evaluate directly the risk of sexual transmission of EVD. RESULTS Of 210 participants who provided an initial semen specimen for analysis, 57 (27%) had positive results on quantitative RT‐PCR. Ebola virus RNA was detected in the semen of all 7 men with a specimen obtained within 3 months after ETU discharge, in 26 of 42 (62%) with a specimen obtained at 4 to 6 months, in 15 of 60 (25%) with a specimen obtained at 7 to 9 months, in 4 of 26 (15%) with a specimen obtained at 10 to 12 months, in 4 of 38 (11%) with a specimen obtained at 13 to 15 months, in 1 of 25 (4%) with a specimen obtained at 16 to 18 months, and in no men with a specimen obtained at 19 months or later. Among the 46 participants with a positive result in phase 1, the median baseline cycle‐threshold values (higher values indicate lower RNA values) for the NP and VP40 targets were lower within 3 months after ETU discharge (32.4 and 31.3, respectively; in 7 men) than at 4 to 6 months (34.3 and 33.1; in 25), at 7 to 9 months (37.4 and 36.6; in 13), and at 10 to 12 months (37.7 and 36.9; in 1). In phase 2, a total of 11 participants had positive results for NP and GP targets (samples obtained at 4.1 to 15.7 months after ETU discharge); cycle‐threshold values ranged from 32.7 to 38.0 for NP and from 31.1 to 37.7 for GP. CONCLUSIONS These data showed the long‐term presence of Ebola virus RNA in semen and declining persistence with increasing time after ETU discharge. (Funded by the World Health Organization and others.)

Differential Virulence of West Nile Strains for American Crows

Increased viremia and deaths in American Crows inoculated with a North American West Nile viral genotype indicate that viral genetic determinants enhance avian pathogenicity and increase transmission potential of WNV.

Arbovirus evolution in vivo is constrained by host alternation

The intrinsic plasticity of RNA viruses can facilitate host range changes that lead to epidemics. However, evolutionary processes promoting cross-species transfers are poorly defined, especially for arthropod-borne viruses (arboviruses). In theory, cross species transfers by arboviruses may be constrained by their alternating infection of disparate hosts, where optimal replication in one host involves a fitness tradeoff for the other. Accordingly, freeing arboviruses from alternate replication via specialization in a single host should accelerate adaptation. This hypothesis has been tested by using cell culture model systems with inconclusive results. Therefore, we tested it using an in vivo system with Venezuelan equine encephalitis virus (VEEV), an emerging alphavirus of the Americas. VEEV serially passaged in mosquitoes exhibited increased mosquito infectivity and vertebrate-specialized strains produced higher viremias. Conversely, alternately passaged VEEV experienced no detectable fitness gains in either host. These results suggest that arbovirus adaptation and evolution is limited by obligate host alternation and predict that arboviral emergence via host range changes may be less frequent than that of single host animal RNA viruses.

Overwintering of West Nile Virus in Southern California

Abstract West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003–2004 and 2004–2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November–March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence in southern California. Continued transmission and vertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.

Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone

Summary The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission.

Evolutionary relationship between Old World West Nile virus strains: Evidence for viral gene flow between africa, the middle east, and europe

Little is known about the genetic relationships between European and other Old-World strains of West Nile virus (WNV) and persistence of WNV North of Mediterranean. We characterized the complete genomes of three WNV strains from France (horse-2000), Tunisia (human-1997) and Kenya (mosquito-1998), and the envelope, NS3 and NS5 genes of the Koutango virus. Phylogenetic analyses including all available full-length sequences showed that: (1) Koutango virus is a distant variant of WNV; (2) the three characterized strains belong to lineage 1, clade 1a; (3) the Tunisian strain roots the lineage of viruses introduced in North America. We established that currently available partial envelope sequences do not generate reliable phylogenies. Accordingly, establishing a large WNV sequence database is pivotal for the understanding of spatial and temporal epidemiology of this virus. For rapid completion of that purpose, colinearized E-NS3-NS5 gene sequences were shown to constitute a valuable surrogate for complete sequences.