Haemovigilance survey and screening strategy for arthropod-borne viruses in blood donors from Argentina.

Arthropod-borne viruses (arboviruses) count among emerging infections, which represent a major challenge for transfusion safety worldwide. To assess the risk of arboviruses-transmission by transfusion (ATT), we performed a survey to evaluate the potential threat for transfusion safety. Samples were retrospectively and randomly collected from donors who donated during the peak of dengue incidence in Cordoba (years: 2016 and 2019-2022). A cost-efficient strategy for molecular screening was implemented with a nucleic acid test (NAT) configured with Flavivirus and Alphavirus-universal degenerated primers targeting conserved gene regions. Besides, we evaluated the neutralizing antibody (NAb) prevalence by plaque reduction neutralization test (PRNT). A total of 1438 samples were collected. Among the NAT-screened samples, one resulted positive for Flavivirus detection. Subsequent sequencing of the PCR product revealed Saint Louis Encephalitis Virus (SLEV) infection (GeneBank accession number OR236721). NAb prevalence was 2.95% for anti-Dengue, 9.94% anti-SLEV, 1.09% anti-West Nile Virus, and 0% anti-Chikungunya. One of the NAb-positive samples also resulted positive for IgM against SLEV but negative by ARN detection. This is the first haemovigilance study developed in Argentina that evaluates the potential risk of ATT and the first research to determine the prevalence of NAb against Flavivirus through PNRT to avoid possible cross-reactions between Ab against Flavivirus. Herein, the finding of one SLEV-viremic donor and the detection of anti-SLEV IgM in a different donor demonstrated a potential threat for transfusion safety and emphasized the need for increased vigilance and proactive measures to ensure the safety of blood supplies.

Acute disseminated encephalomyelitis following Saint Louis encephalitis virus infection.

Saint Louis encephalitis virus (SLEV) infection is an arbovirosis associated with a broad spectrum of neurological complications. We present a case of a 55-year-old man hailing from Manaus, a city situated in the heart of the Amazon Rainforest, who exhibited symptoms of vertigo, tremors, urinary and fecal retention, compromised gait, and encephalopathy 3 weeks following SLEV infection. Neuroaxis MRI revealed diffuse, asymmetric, and poorly defined margins hyperintense lesions with peripheral and ring enhancement in subcortical white matter, as well as severe spinal cord involvement. Serology for SLEV was positive both on serum and cerebrospinal fluid. To the best of our knowledge, the present report is the first to show brain lesions along with myelitis as a post-infectious complication of SLEV infection.

Effect of Agroecosystems on Seroprevalence of St. Louis Encephalitis and West Nile Viruses in Birds, La Pampa, Argentina, 2017-2019.

In Argentina, the Pampa ecoregion has been almost completely transformed into agroecosystems. To evaluate the environmental (agricultural area, tree coverage, distance to the nearest water body and urban site) and biological (dove, cowbird, and sparrow abundance) effects on free-ranging bird exposure to St. Louis encephalitis virus (SLEV) and West Nile virus (WNV), we used generalized linear mixed models. For 1,019 birds sampled during 2017-2019, neutralizing antibodies were found against SLEV in samples from 60 (5.8%) birds and against WNV for 21 (2.1%). The best variable for explaining SLEV seroprevalence was agricultural area, which had a positive effect; however, for WNV, no model was conclusive. Our results suggest that agroecosystems in the La Pampa ecoregion increase the exposure of avian hosts to SLEV, thus potentially increasing virus activity.

Detection of Saint Louis encephalitis virus in two Brazilian states.

We describe the circulation of Saint Louis encephalitis virus (SLEV) in two Brazilian States during outbreaks of Dengue and Zika viruses. We detected the virus in a patient from Araraquara, State of São Paulo, and in patients and in a mosquito pool of Culex quinquefasciatus from Sinop, State of Mato Grosso. Phylogenetic analysis grouped samples from this study within genotype V, which are closely related to other strains that previously circulated in other parts of the country. Genotype V seems to have established circulation in Brazil.

Arbovirus serosurvey (Orthobunyavirus, Flavivirus, and Alphavirus) in a draft horse population from Santa Fe, Argentina (2013-2016).

In the present study, we serosurveyed the exposure of 222 draft horses to different arboviruses in the city of Santa Fe, Argentina. Plaque reduction neutralization tests confirmed exposure to Fort Sherman virus (FSV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), and Río Negro virus (RNV). Apparently, Western and Eastern equine encephalitis viruses did not circulate in the population tested. The confirmation of five seroconversions for WNV, FSV, and SLEV and the association between prevalence and age are indicative of recent circulation. These results highlight the importance of considering draft horses in arboviral surveillance in urban and rural areas of developing countries.

Autochthonous circulation of Saint Louis encephalitis and West Nile viruses in the Province of La Rioja, Argentina.

St. Louis encephalitis (SLEV) and West Nile (WNV) arboviruses, which circulate in Argentina, are maintained in enzootic transmission cycles involving Culex mosquitoes (vectors) and birds belonging to orders Passeriformes and Columbiformes (amplifier hosts). The objective of this work was to determine the circulation of both viruses among wild birds in a semiarid ecosystem in the Province of La Rioja through a serologic survey. During spring 2013 and fall 2014, a total of 326 wild birds belonging to 41 species were captured in areas close to the cities of La Rioja and Chilecito, in the Province of La Rioja. While exposure to SLEV and WNV was analyzed in birds' serum through neutralizing antibody detection, viral circulation was estimated through apparent seroprevalence of neutralizing antibodies. The exposure of the avian community to viruses was 3.02% for SLEV and 1.89% for WNV, while 1.19% corresponded to coinfections. Our study confirms for the first time the circulation of SLEV and WNV in wild birds in the Province of La Rioja. Moreover, it is the first study to register neutralizing antibodies for flavivirus in the species Leptotila verreauxi (White-tipped Dove) (WNV) and Melanerpes cactorum (White-fronted Woodpecker) (SLEV). These results suggest that in semiarid ecosystems from northwestern Argentina the requirements and conditions for amplification and enzootic maintenance of SLEV and WNV would be present.

Specific diagnostic method for St. Louis encephalitis virus using a non-structural protein as the antigen.

St. Louis encephalitis virus (SLEV) is a mosquito-borne re-emerging flavivirus in Argentina. It is currently necessary to develop specific serological tests that can efficiently discriminate the flaviviruses that circulate in our country. The immunoassays to diagnose SLEV lack specificity because they are based on the detection of structural viral proteins and the human immunoglobulins produced during infection against these proteins cross-react with other flaviviruses. Here, we describe an enzyme-immunoassay designed to detect human IgG antibodies specific to the viral non-structural protein NS5. The results indicate that NS5 is a promising antigen useful to discriminate SLEV from other circulating flaviviruses.

Serological survey for Saint Louis encephalitis virus and West Nile virus in domestic mammals in Córdoba, Argentina: are our pets potential sentinels?

We evaluated the seroprevalence of Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) in dogs and cats in Córdoba, Argentina. Monotypic and heterotypic serological patterns were differentiated by means of a neutralization test. The SLEV seroprevalence in dogs was 14.6% (44/302; 100% monotypic). Two out of 94 (2.1%, 100% monotypic) cats were positive for WNV only. Four dogs (1.3%) exhibited neutralizing antibody titers against SLEV and WNV. During the study, three dogs seroconverted to SLEV. Our study demonstrates that pets were useful for detecting viral activity and could be considered as sentinels in the local surveillance of SLEV and WNV.

Assessment of Culex pipiens bioforms in the world's southernmost distribution limit.

The mosquito Culex pipiens s.s. L. occurs as two bioforms that differ in physiology and behaviour affecting virus transmission cycles. To assess the occurrence of Cx. pipiens bioforms in the southernmost limit of its distribution, specimens were collected aboveground in southern Buenos Aires Province and east Patagonia, Argentina. Ten larvae and 25 adults were individually processed and identified by polymerase chain reaction (PCR) amplification of Ace-2 and CQ11 loci. Culex quinquefasciatus Say (one larva, two adults), Cx. pipiens f. molestus (one larva, one adult) and one adult of hybrid origin were identified in Buenos Aires Province; only Cx. pipiens f. molestus was recorded in Patagonia (eight larvae, 21 adults). The potential absence of bioform pipiens and its implications in arbovirus enzootic cycles is discussed.

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis.

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Detection of antibodies against Icoaraci, Ilhéus, and Saint Louis Encephalitis arboviruses during yellow fever monitoring surveillance in non-human primates (Alouatta caraya) in southern Brazil.

BACKGROUND: Free-ranging non-human primates (NHPs) can host a variety of pathogenic microorganisms, such as arboviruses, which include the yellow fever virus (YFV). This study aimed to detect the circulation of YF and other arboviruses in three wild Alouatta caraya populations in forests in southern Brazil. METHODS: We collected 40 blood and serum samples from 26 monkeys captured/recaptured up to four times from 2014 to 2016, searching for evidence of arboviruses by virus isolation, PCR, and neutralization tests. RESULTS: Viral isolation and genome detection were negative; however, we detected neutralizing antibodies against the Saint Louis, Ilhéus, and Icoaraci viruses in three NHPs. CONCLUSIONS: Saint Louis Encephalitis, Ilhéus, and Icoaraci viruses circulated recently in the region. Future studies should investigate the role of NHPs, other vertebrate hosts and wild vectors in the region's arbovirus circulation and the potential risks of the arboviruses to wildlife, domestic animals, and humans.

Reemergence of St. Louis Encephalitis Virus in the Americas.

We summarize and analyze historical and current data regarding the reemergence of St. Louis encephalitis virus (SLEV; genus Flavivirus) in the Americas. Historically, SLEV caused encephalitis outbreaks in the United States; however, it was not considered a public health concern in the rest of the Americas. After the introduction of West Nile virus in 1999, activity of SLEV decreased considerably in the United States. During 2014-2015, SLEV caused a human outbreak in Arizona and caused isolated human cases in California in 2016 and 2017. Phylogenetic analyses indicate that the emerging SLEV in the western United States is related to the epidemic strains isolated during a human encephalitis outbreak in Córdoba, Argentina, in 2005. Ecoepidemiologic studies suggest that the emergence of SLEV in Argentina was caused by the introduction of a more pathogenic strain and increasing populations of the eared dove (amplifying host).

Diagnosis of Fatal Human Case of St. Louis Encephalitis Virus Infection by Metagenomic Sequencing, California, 2016.

We used unbiased metagenomic next-generation sequencing to diagnose a fatal case of meningoencephalitis caused by St. Louis encephalitis virus in a patient from California in September 2016. This case is associated with the recent 2015-2016 reemergence of this virus in the southwestern United States.

Neuroinvasive St. Louis Encephalitis Virus Infection in Solid Organ Transplant Recipients.

In summer 2015, three unrelated solid organ transplant recipients in Phoenix, Arizona, had meningoencephalitis suggestive of West Nile virus (WNV) infection. Testing was inconclusive but was later confirmed as St. Louis encephalitis (SLE). We retrospectively reviewed clinical manifestations, treatment, and outcomes of these transplant recipients. Common symptoms were fever, rigors, diarrhea, headache, and confusion. One patient died 3 days after hospitalization. Therapy for the other two patients was initiated with interferon α-2b (IFN) and intravenous IgG (IVIG; IFN plus IVIG in combination). Both patients tested positive for WNV by serologic assay, but SLE virus (SLEV) infection was later confirmed by plaque reduction neutralization test at a reference laboratory. Clinical improvement was observed within 72 h after initiation of IFN plus IVIG. SLEV has been an uncommon cause of neuroinvasive disease in the United States. Accurate, timely diagnosis is hindered because of clinical presentation similar to neuroinvasive WNV and SLE, serologic cross-reactivity, and lack of a commercially available serologic assay for SLEV. There is currently no approved therapy for flaviviral neuroinvasive disease. Anecdotal reports indicate varying success with IFN, IVIG, or IFN plus IVIG in WNV neuroinvasive disease. The same regimen might be of value for immunocompromised persons with neuroinvasive SLEV infection.

Mutagen resistance and mutation restriction of St. Louis encephalitis virus.

The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus, is a mosquito-borne flavivirus that has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modelling of the E416K polymerase mutation indicated its location in the pinky finger domain of the RdRp, distant from the active site. Deep sequencing of the E416K mutant revealed lower genetic diversity than wild-type SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed that E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with West Nile virus high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference response, suggesting an impaired capacity to escape RNA interference could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and give further insight into the consequences of altered fidelity of flaviviruses.

Development of a model of Saint Louis encephalitis infection and disease in mice.

BACKGROUND: Flaviviruses are a genre of closely related viral pathogens which emerged in the last decades in Brazil and in the world. Saint (St.) Louis encephalitis virus (SLEV) is a neglected flavivirus that can cause a severe neurological disease that may lead to death or sequelae. St. Louis encephalitis pathogenesis is poorly understood, which hinders the development of specific treatment or vaccine. METHODS: To address this problem, we developed a model of SLEV infection in mice to study mechanisms involved in the pathogenesis of severe disease. The model consists in the intracranial inoculation of the SLEV strain BeH 355964, a strain isolated from a symptomatic human patient in Brazil, in adult immunocompetent mice. RESULTS: Inoculated mice presented SLEV replication in the brain, accompanied by tissue damage, disease signs, and mortality approximately 7 days post infection. Infection was characterized by the production of proinflammatory cytokines and interferons and by leukocyte recruitment to the brain, composed mainly by neutrophils and lymphocytes. In vitro experiments indicated that SLEV is able to replicate in both neurons and glia and caused neuronal death and cytokine production, respectively. CONCLUSIONS: Altogether, intracranial SLEV infection leads to meningoencephalitis in mice, recapitulating several aspects of St. Louis encephalitis in humans. Our study indicates that the central nervous system (CNS) inflammation is a major component of SLEV-induced disease. This model may be useful to identify mechanisms of disease pathogenesis or resistance to SLEV infection.

St. Louis encephalitis virus possibly transmitted through blood transfusion-Arizona, 2015.

BACKGROUND: St. Louis encephalitis virus is a mosquito-borne flavivirus that infrequently causes epidemic central nervous system infections. In the United States, blood donors are not screened for St. Louis encephalitis virus infection, and transmission through blood transfusion has not been reported. During September 2015, St. Louis encephalitis virus infection was confirmed in an Arizona kidney transplant recipient. An investigation was initiated to determine the infection source. STUDY DESIGN AND METHODS: The patient was interviewed, and medical records were reviewed. To determine the likelihood of mosquito-borne infection, mosquito surveillance data collected at patient and blood donor residences in timeframes consistent with their possible exposure periods were reviewed. To investigate other routes of exposure, organ and blood donor and recipient specimens were obtained and tested for evidence of St. Louis encephalitis virus infection. RESULTS: The patient presented with symptoms of central nervous system infection. Recent St. Louis encephalitis virus infection was serologically confirmed. The organ donor and three other organ recipients showed no laboratory or clinical evidence of St. Louis encephalitis virus infection. Among four donors of blood products received by the patient via transfusion, one donor had a serologically confirmed, recent St. Louis encephalitis virus infection. Exposure to an infected mosquito was unlikely based on the patient's minimal outdoor exposure. In addition, no St. Louis encephalitis virus-infected mosquito pools were identified around the patient's residence. CONCLUSION: This investigation provides evidence of the first reported possible case of St. Louis encephalitis virus transmission through blood product transfusion. Health care providers and public health professionals should maintain heightened awareness for St. Louis encephalitis virus transmission through blood transfusion in settings where outbreaks are identified.

Reemergence of St. Louis Encephalitis Virus, California, 2015.

St. Louis encephalitis virus infection was detected in summer 2015 in southern California after an 11-year absence, concomitant with an Arizona outbreak. Sequence comparisons showed close identity of California and Arizona isolates with 2005 Argentine isolates, suggesting introduction from South America and underscoring the value of continued arbovirus surveillance.

Concurrent Outbreaks of St. Louis Encephalitis Virus and West Nile Virus Disease - Arizona, 2015.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that can cause outbreaks of acute febrile illness and neurologic disease. Both viruses are endemic throughout much of the United States and have the same Culex species mosquito vectors and avian hosts (1); however, since WNV was first identified in the United States in 1999, SLEV disease incidence has been substantially lower than WNV disease incidence, and no outbreaks involving the two viruses circulating in the same location at the same time have been identified. Currently, there is a commercially available laboratory test for diagnosis of acute WNV infection, but there is no commercially available SLEV test, and all SLEV testing must be performed at public health laboratories. In addition, because antibodies against SLEV and WNV can cross-react on standard diagnostic tests, confirmatory neutralizing antibody testing at public health laboratories is usually required to determine the flavivirus species (2). This report describes the first known concurrent outbreaks of SLEV and WNV disease in the United States.

First genome sequence of St. Louis encephalitis virus (SLEV) isolated from a human in Brazil.

St. Louis encephalitis virus (SLEV), a member of the family Flaviviridae, genus Flavivirus, is a causative agent of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the first outbreak of SLEV in Brazil was identified only in 2007, concomitant with an outbreak of dengue virus (DENV) serotype 3. This finding, along with other reports, indicates that SLEV circulation in Brazil is largely unknown, and there may be epidemiological implications of the co-circulation of SLEV, DENV and other flaviviruses in Brazil. Here, we describe the first complete genome sequence of an SLEV strain isolated from a human patient in Brazil, strain BeH 355964. Phylogenetic analysis was performed to determine the genotype of BeH 355964 using the full-length genome and envelope (E) gene sequences separately. Both analyses showed that BeH 355964 could be classified as genotype V. Although the number of single gene sequences available is greater (such as for the E gene), the phylogenetic tree based on the complete genome sequence was better supported and provided further information about the virus.