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(Re-)emerging arboviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Usutu virus (USUV), are continuously increasing in incidence. We analyzed the epidemiological characteristics of flavivirus infections in humans, sentinel animals, and mosquitoes detected in the 2022 transmission season in Croatia. From April to November 2022, 110 hospitalized patients with neuroinvasive diseases (NID) were tested for the presence of arboviruses. RT-qPCR was used to detect TBEV, WNV, and USUV RNA. An ELISA and virus neutralization tests were used for the detection of flavivirus antibodies. TBEV infection was confirmed in 22 patients with NID. WNV NID was detected in six patients. TBE showed male predominance (81.8%; male-to-female ratio of 4.5:1). All but one WNV patients were males. TBE occurred from April to August, with the majority of patients (83.3%) being detected during the May–June–July period. WNV infections were recorded in August and September. In addition to human cases, asymptomatic WNV infections (IgM positive) were reported in 10 horses. For the first time in Croatia, WNV NID was observed in one horse that presented with neurological symptoms. Furthermore, USUV was confirmed in one dead blackbird that presented with neurological symptoms. A total of 1984 mosquitoes were collected in the City of Zagreb. Two Ae. albopictus pools tested positive for flavivirus RNA: one collected in July (USUV) and the other collected in August (WNV). A phylogenetic analysis of detected human and avian strains confirmed WNV lineage 2 and the USUV Europe 2 lineage. The presented results confirm the endemic presence of neuroinvasive flaviviruses in continental Croatia. The continuous monitoring of virus circulation in humans, sentinel animals, and mosquitoes is needed to reduce the disease burden.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are the most important neuroinvasive arboviruses detected in Europe. In this study, we analyzed cerebrospinal fluid (CSF) concentrations of 12 proinflammatory chemokines (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11) in 77 patients with neuroinvasive diseases (NIDs). Flavivirus infection was confirmed in 62 patients (TBEV and WNV in 31 patients each), while in 15 patients the etiology of NID was not determined (NDE). Similar patterns of high-level expression of chemokines regulating monocyte/macrophage responses (CCL2), neutrophil recruitment (CXCL1 and CXCL8), and interferon-inducible chemoattractants for leukocytes (CXCL10 and CXCL11) have been observed in WNV and TBEV groups. None of the tested chemokines significantly differed between patients with TBEV or WNV. Concentrations of CCL17, CCL20, CXCL5, CXCL10, and CXCL11 were significantly lower in both WNV and TBEV groups compared to NID NDE patients. The logistic regression model showed that CSF concentrations of CXCL11, CXCL5, and CXCL10 could potentially be used for the classification of patients into the WNV or TBEV group versus groups with other NIDs. This study identified, for the first time, similar patterns of CSF chemokine expression in WNV and TBEV infections, suggesting common immunopathogenic mechanisms in neuroinvasive flavivirus infections that should be further evaluated.

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks before the disease onset. High titers of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) were found in both serum and cerebrospinal fluid (CSF) samples, indicating acute TBEV infection. West Nile virus (WNV) and Usutu virus (USUV) IgM and/or IgG antibodies were also detected, and a virus neutralization test (VNT) was performed. A high titer of TBEV neutralizing (NT) antibodies (640) was detected, which confirmed acute TBE. However, NT antibodies to WNV and USUV were also detected (titer 80 for both viruses). After TBEV and WNV IgG avidity evaluation, previous flavivirus infection was highly suspected (avidity index 82% and 89%, respectively). Blood, CSF, and urine samples were negative for respective viruses’ RNA. The presented case highlights the challenges in flavivirus serodiagnosis. In the published literature, different degrees of cross-reactivity or cross-neutralization between TBEV and dengue, louping ill, Omsk hemorrhagic fever, Langat, and Powassan virus were also observed. Therefore, the serology results should be interpreted with caution, including the possibility of cross-reactivity. In areas where several flaviviruses co-circulate VNT is recommended for disease confirmation.

The epidemiology of West Nile (WNV) and Usutu virus (USUV) has changed dramatically over the past two decades. Since 1999, there have been regular reports of WNV outbreaks and the virus has expanded its area of circulation in many Southern European countries. After emerging in Italy in 1996, USUV has spread to other countries causing mortality in several bird species. In 2009, USUV seroconversion in horses was reported in Italy. Co-circulation of both viruses was detected in humans, horses and birds. The main vector of WNV and USUV in Europe is Culex pipiens, however, both viruses were found in native Culex mosquito species (Cx. modestus, Cx. perexiguus). Experimental competence to transmit the WNV was also proven for native and invasive mosquitoes of Aedes and Culex genera (Ae. albopictus, Ae. detritus, Cx. torrentium). Recently, Ae. albopictus and Ae. japonicus naturally-infected with USUV were reported. While neuroinvasive human WNV infections are well-documented, USUV infections are sporadically detected. However, there is increasing evidence of a role of USUV in human disease. Seroepidemiological studies showed that USUV circulation is more common than WNV in some endemic regions. Recent data showed that WNV strains detected in humans, horses, birds, and mosquitoes mainly belong to lineage 2. In addition to European USUV lineages, some reports indicate the presence of African USUV lineages as well. The trends in WNV/USUV range and vector expansion are likely to continue in future years. This mini-review provides an update on the epidemiology of WNV and USUV infections in Southern Europe within a multidisciplinary “One Health” context.

West Nile virus (WNV) is one of the most widely distributed (re-)emerging arboviruses. In Croatia, acute WNV infections as well as seropositivity were detected in humans, horses, birds and poultry. Although serologic evidence of WNV human infections dates back to the 1970s, no clinical cases were reported until 2012. WNV outbreaks, as well as sporadic infections, were continuously recorded in continental Croatian counties from 2012 to 2018. In addition, acute asymptomatic infections (IgM antibodies) in horses have been regularly notified in continental regions since 2012, while seropositive horses (seroprevalence rates 3.7–21.4%) were detected in both continental and coastal regions. Moreover, WNV seropositivity in poultry (1.8–22.9%) was reported from 2013 to 2020. During the largest WNV outbreak in 2018, WNV RNA was detected for the first time in two dead goshawks (Accipiter gentilis) from the same aviary in North-West Croatia, while WNV antibodies were found in one buzzard (Butteo butteo) from the same region. In addition, WNV RNA was detected in a dead blackbird (Turdus merula) at the Croatian littoral. The phylogenetic analysis of 11 strains detected in urine samples of patients with neuroinvasive disease and 1 strain detected in a goshawk showed circulation of WNV lineage 2. Thus far, WNV has not been detected in mosquitoes in Croatia.

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

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 < 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.

Abstract 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.