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In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re-occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real-time RT- PCR. Positive samples were tested by nested RT- PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA with confirmation of cross- reactive samples by virus neutralization test (VNT). WNV neuroinvasive disease was confirmed in 54 and WNV fever in seven patients from 10 continental Croatian counties. Areas affected in 2018 were those in which cases occurred in previous seasons, while in three areas human cases were reported for the first time. Phylogenetic analysis of six strains from patients residing in different geographic areas showed circulation of WNV lineage 2. In three patients, neuroinvasive USUV infection was confirmed by RT-PCR or VNT. Sequence analysis of one detected strain revealed USUV Europe 2 lineage. During the same period, a total of 2, 574 horse and 1, 069 poultry serum samples were tested for WNV antibodies using ELISA. Acute asymptomatic WNV infection (IgM antibodies) was documented in 20/0.7% horses. WNV IgG antibodies were found in 307/11.9% horses and in 125/12.7% poultry. WNV RNA was detected in two goshawks and USUV RNA was detected in one blackbird from north-western Croatia. In the Zagreb area, 3, 670 female mosquitoes were collected. One Culex pipiens pool collected in July tested positive for USUV RNA. Our results highlight the importance of continuous multidisciplinary 'One health' surveillance of these emerging arboviruses.

Cardiac involvement has rarely been reported in West Nile (WNV) infection. We report a fatal case of WNV encephalitis associated with an acute anteroseptal ST elevation myocardial infarction. The patient was hospitalized with a fever, headache, nausea and vomiting. The physical examination revealed positive meningeal signs and an altered level of consciousness. High levels of cardiac enzymes (creatine phosphokinase/MB fraction, lactate dehydrogenase, myoglobin and cardiac troponin I) and ST elevation on electrocardiogram were found. Both CSF and urine samples were positive for WNV RNA. This case highlights the need of awareness of the possibility of a WNV-related myocardial infection, including myocardial infarction.

The West Nile virus (WNV) is a widely distributed emerging mosquito-borne arbovirus. Although the majority of WNV infections are asymptomatic (80%), some infected individuals (20%) present with non- specific febrile disease (WNV fever) and less than 1% develop neuroinvasive diseases (meningitis, encephalitis, and myelitis). Several atypical or rare presentations of WNV infection such as cerebellitis, myocarditis, hepatitis, pancreatitis, and rhabdomyolysis have been described in case reports or small case series1. Although rare, ocular manifestations, including monofocal or multifocal WNV chorioretinitis are the most commonly reported clinical manifestations of WNV infection after fever and neuroinvasive disease.

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50, serum median 24.48, IQR 11.93–49.81, p &lt, 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.

INTRODUCTION: West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. ----- METHODS: Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. ----- RESULTS: WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). ----- CONCLUSIONS: Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.

Purpose: To present a case of acute anteroseptal ST-elevation myocardial infarction in the West Nile virus (WNV) meningoencephalitis. Methods & Materials: A 77- year-old patient was hospitalized in the late summer of 2017 on the second day of the illness manifested by a fever up to 38.6 °C and diarrhea. The patient did not report recent travel but recalled mosquito bites. Past medical history included hypertension. At admission, routine laboratory tests, electrocardiogram (ECG) and chest x-ray were performed. In addition, cerebrospinal fluid (CSF), urine and blood samples were collected for a virological analysis. Results: At admission, WBC count was 24.6 (reference range 3.4-9.7x10(9)/L) with neutrophilia (92%, range 44-72%) and very high levels of cardiac enzymes: creatinine phosphokinase 1856 (range 17-153 U/I), lactate dehydrogenase 433 (range 2-241 U/L), myoglobin 3116 (range 20-80 ug/L) and troponin I 17.640 (range 0.000-0.056 ug/L). ECG showed ST elevation. In the cardiac intensive care unit, an emergency coronary angiography was performed which confirmed the coronary artery stenosis. The patient's condition complicated on the 4th day of the illness by an altered level of consciousness with progression to coma, accompanied by neck stiffness and positive meningeal signs. Computed tomography of the brain was normal. Cerebrospinal fluid (CSF) showed pleocytosis with 26 cells/mm3, predominantly mononuclears (73%) and elevated protein level (1.151, range 0.170-0.370 g/L). Both CSF and urine were positive for WNV RNA by real- time and nested RT-PCR. Phylogenetic analysis showed WNV lineage 2. The patient was initially treated with acyclovir, ampicillin and cefepime parenterally with supportive therapy (antiedematous, antiaggregation and antihypertensive therapy). On the 8th day of the illness a respiratory insufficiency developed. The patient was intubated and mechanically ventilated, but developed hypotension and low oxygen saturation in spite of an adequate respiratory support. Despite the cardiopulmonary resuscitation, the patient died due to cardiopulmonary arrest. Conclusion: Although cardiac involvement is not frequently reported in the course of a WNV infection, physicians should be aware of the possibility of a WNV-related myocardial infection.