**1. Introduction**

West Nile virus (WNV) is a mosquito-borne arbovirus belonging to the family *Flaviviridae*, genus *Flavivirus*, Japanese encephalitis serocomplex. In nature, it circulates mainly among birds and mosquitoes of genera *Culex*, *Anopheles*, *Culiseta*, *Uranotaenia* or *Coquilletidia*. WNV is a causative agen<sup>t</sup> of West Nile fever, a mosquito-borne disease affecting horses and humans, the latter serving as so-called dead-end hosts [1]. Since 2004, highly virulent WNV lineage 2 has appeared in Europe, causing sporadic outbreaks in Hungary (2008), Greece (2010) and Serbia (2012) [2]. In the Czech Republic, WNV research in birds, mosquito vectors and humans has a long tradition. Importantly, highly virulent WNV lineage 2 (WNV-2) strains have been repeatedly documented in *Cx. modestus* populations on local fishponds [3,4]. Interestingly, no case of WNV-2 infection in humans had been documented before the 2018 season.

**Citation:** Zelená, H.; Kleinerová, J.; Šikutová, S.; Straková, P.; Kocourková, H.; Stebel, R.; Husa, P.; Husa, P., Jr.; Tesaˇrová, E.; Lejdarová, H.; et al. First Autochthonous West Nile Lineage 2 and Usutu Virus Infections in Humans, July to October 2018, Czech Republic. *Pathogens* **2021**, *10*, 651. https://doi.org/10.3390/ pathogens10060651

Academic Editor: Francisco Llorente

Received: 28 April 2021 Accepted: 21 May 2021 Published: 24 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Usutu virus is a mosquito-borne flavivirus (family *Flaviviridae*, genus *Flavivirus*), in the Japanese encephalitis virus serocomplex. Like WNV, it circulates among birds and ornithophilic mosquitoes, with demonstrated pathogenicity to a wide variety of wild and domestic birds. It emerged in 2001 in Austria with the highest mortality recorded in blackbirds [5]. Since then USUV has spread further to central and western European countries including the Czech Republic, where it has established itself among blackbirds [6,7] and mosquitoes [8]. Since its introduction in Europe, there have been several reports of neuroinvasive disease in immunocompromised [9] and immunocompetent humans [10,11]. Similar to WNV, USUV has been detected in asymptomatic blood donors in Italy [12], Austria [13] and Germany [14].

The aim of this report was to summarize the epidemiological and clinical characteristics of patients diagnosed by infection with either WNV-2 or USUV in the Czech Republic and to expand our knowledge of these emerging mosquito-borne diseases in Central Europe.

### **2. Case Reports**

We summarized epidemiological, clinical and laboratory findings collected from five patients diagnosed with WNV or USUV infections. Specific data are summarized in Tables 1–3. All but one had no history of traveling to a known WNV endemic area. One patient (Case 1) reported previous vaccination against tick-borne encephalitis virus (TBEV), a related tick-borne flavivirus circulating in the area, that might present with cross-reactive immunity, which could subsequently complicate an accurate final diagnostic result. All patients reside in South Moravia.

**Table 1.** Descriptive epidemiological and clinical summary of five patients diagnosed with mosquito-borne disease, from July to October 2018, Czech Republic.


Legend: TBE-Tick-borne encephalitis; YF-Yellow fever; GCS-Glasgow Coma Scale; GOS-Glasgow Outcome Scale; NA—not applicable.


**Table 2.** Laboratory and neuropathological findings of five patients diagnosed with mosquito-borne disease, from July to October 2018, Czech Republic.


*Pathogens* **2021**, *10*, 651

**Table 3.** Diagnostic outcomes of five patients diagnosed with mosquito-borne disease, from July to October 2018, Czech Republic.


California, U.S. (Cat Nr EL 0300 G,M); 3 West Nile Virus IgG, IgM IIFT, Euroimmun, Lübeck, Germany (Cat Nr. FI 2662-1005 G,M); 4 ELISA-Viditest anti-TBEV IgG, IgG avidity and IgM,

Vidia, Vestec, Czech Republic (Cat Nr. OD-170, OD-194).

**Table 3.** *Cont.*

### *2.1. Description of Cases*

### 2.1.1. Case 1

On 3 August 2018, a 74-year-old man was examined at the Infectious Diseases Clinic of Brno University Hospital for fever up to 40 ◦C for 12 days. Fever was accompanied by flu-like symptoms and severe headaches. The patient reported diarrhea during the first 4 days and observed macular rash on the skin on the 6th day after the onset of the fever. Apart from dehydration with borderline hypotension, the clinical examination revealed no obvious pathology. The patient had no symptoms of meningitis; no cerebrospinal fluid was tested. Basic laboratory samples were taken as early as 31 July 2018 (see Table 2). No increase in inflammatory parameters, no leukocytosis and no other pathology was found. Treatment was symptomatic with antipyretics, analgesics and hydration by infusion; there was no antibiotic therapy. A clinical follow-up was performed after 4 days, rash and fever subsided and temperatures also decreased after 13 days. Exhaustion and occasional headache persisted. Further stool and urine cultures were negative, as were CMV serology and stool virology. Inflammatory parameters remained low. In order to exclude infectious foci, a number of additional examinations were performed (chest X-ray, abdominal and intestinal ultrasound, dentistry, echocardiography). The only significant finding was tooth decay; the dentist recommended the extraction of several teeth over time, but ruled out the odontogenic etiology of the fever. The patient's exhaustion and fatigue slowly disappeared, the fever did not return, and recovery lasted approximately 7 weeks. The man was not hospitalized; he was in the home care of his wife—a doctor. The patient gradually returned to regular activities, including recreational sport. The diagnosis of West Nile Fever was made ex post. The serological finding of WNV correlated with the protracted clinical course of the febrile viral disease.

**Epidemiological background:** The patient's place of residence is metropolitan Brno. From June 2018 the patient slept outside on the covered terrace of a family villa. The patient stayed in Brno—except for a day trip on 6 May 2018 to the aqua park in Laa an der Thaya (Austria). From 31 May 2018 to 20 July 2018 the patient spent a total of 10 days in a cottage by a pond north of Brno. The patient was vaccinated against TBEV.

### 2.1.2. Case 2

The second case report concerns a 72-year-old female patient admitted on 13 August 2018 at the internal department of the Bˇreclav Hospital for general weakness, diarrhea and myalgia lasting 6 days. She was a polymorbid patient treated for rheumatoid arthritis, lichen planus, chronic gastritis and thyroiditis. On the day of admission, the patient was found at home and could not move. She had the status of odontogenic etiology of fever in the internal medicine department. The patient gradually deteriorated, and a quantitative impairment of consciousness appeared on the 6th day of hospitalization (18 August 2018). The patient was transported to the department of anesthesiology, intensive care medicine and resuscitation where she was promptly intubated, artificial sleep was induced and artificial lung ventilation was started. Computed tomography (CT) of the brain showed non-specific hypodense areas in brain tissue. Furthermore, lumbar puncture was supplemented as part of differential diagnosis of the disturbance of consciousness. Biochemical and cytological findings in cerebrospinal fluid corresponded to aseptic neuroinfection. Serology showed borderline IgM and weakly positive IgG antibodies against tick-borne meningoencephalitis in both blood and cerebrospinal fluid. Despite the established intensive therapy, the patient further deteriorated and died on 27 August 2018 with a clinical picture of refractory failure of multiple organs. The suspicion of West Nile virus infection arose only post mortem, after consultation with an infectiologist; additional serological and molecular examinations then confirmed a WNV infection.

**Epidemiological background:** The first epidemiological inquiry was realized relatively soon after the patient´s death and focused primarily on environmental conditions at the place of residence. There were several barrels and tanks filled with stagnant water at the sites, which served as a suitable attractant for mosquitoes. Based on these findings,

several CO2 mosquito traps (Bioquip, Rancho Dominguez U.S.) were installed on 2 capture nights. A total of 87 female mosquitoes belonging to *Cx. pipiens*, *Anopheles maculipennis* sensu lato, *Aedes vexans*, *Ae. caspius* and *Ae. sticticus* were captured and examined for the presence of WNV. No positive sample was found. The patient did not leave the residence. Mosquitoes bit her regularly. She was not vaccinated against TBEV and did not undergo blood transfusion.

### 2.1.3. Case 3

The 51-year-old man was admitted on 2 October 2018 to the Infectious Diseases Department of Bˇreclav Hospital for non-specific symptoms lasting from 9 September 2018—fever, headaches, fatigue, arthralgia and sleep disorders. This was a patient without significant comorbidities, who in the past was treated for Lyme disease and vertebrogenic algic syndrome. As part of the diagnosis, a lumbar puncture was added; biochemical and cytological tests of cerebrospinal fluid corresponded to aseptic neuroinfection. A CT scan of the brain showed no recent pathological changes. Tick-borne meningoencephalitis and herpesvirus or enterovirus neuroinfections were considered for differential diagnosis, but microbiological tests were negative for all these agents. The infectiologist also indicated that serological diagnosis of West Nile fever will be performed at the National Reference Laboratory (NRL) for arboviruses (Public Health Institute, Ostrava). Additional laboratory tests then confirmed a WNV infection. The clinical course of the disease was mild in this case; there were no complications during hospitalization, and the patient was discharged home after 14 days without a residual neurological deficit.

**Epidemiological background:** The epidemiological examination was performed soon after the patient returned from the hospital. The patient lives in a family house with garden and small natural pond; in some places there are barrels of stagnant water. He did not travel outside his residence, except for regular walks in the nearby forest. He denied vaccination against TBEV as well as blood donation/transfusion.

### 2.1.4. Case 4

A 52-year-old man was admitted on 18 September 2018 to the Infectious Diseases Department of the Bˇreclav Hospital for non-specific symptoms lasting 8 days. At first, there were high fever, fatigue, arthralgia and myalgia, and the patient also reported headaches, diarrhea and vomiting during the past 4 days. The patient's medical history included arterial hypertension and long-term headaches of unclear origin. In the department of infectious diseases, lumbar puncture was added; biochemical and cytological tests of cerebrospinal fluid corresponded to aseptic neuroinfection. Due to the characteristics of the complaint, a CT scan of the brain was added with a finding of incipient cerebral oedema. However, later magnetic resonance imaging (MRI) of the brain did not show any intracranial inflammatory changes. The results of routine serological tests (including serology in tick-borne meningoencephalitis) were not convincing. In the light of previous experience (see Case reports 2 and 3), biological material was sent to the NRL for arboviruses for the diagnosis of West Nile fever. The laboratory also confirmed WNV infection in this case. In this case the hospitalization lasted 17 days, there were no complications, and the patient was discharged home in good condition.

**Epidemiological backgound:** Epidemiological investigation was realized by phone call. Patient lives in a family house with garden on the margin of town, close to a fishpond accompanied with rich vegetation. In the garden, several objects (barrels, watering cans) with stagnant water were present. He did not travel outside the region. He denied vaccination to TBEV as well as blood donation/transfusion.

### 2.1.5. Case 5

A 46-year-old woman without a serious previous illness was admitted to the Infectious Diseases Clinic of Brno University Hospital on suspicion of meningoencephalitis on 10 January 2018. A 10-day tourist stay in Turkey preceded the symptoms, but the development

of the symptoms occurred on the 3rd day after leaving for Turkey from the Czech Republic. First, a striking fatigue developed, with the development of headache in the following days and accompanied with nausea and vomiting, vertigo, and later the family observed bradypsichia and dysarthria. She had chills but did not measure her temperature. After admission, the patient was afebrile, stable in terms of the circulatory system, stupor, bradypsichia, still oriented in all qualities, with positive upper meningeal phenomena in the neurostat, fine tremor of the upper extremities and dysmetria, ataxia, standing titubation without lateral predilection. Other objective findings were remarkable (see Table 2). Only a slight increase in CRP was observed in the laboratory analysis; the other biochemical and haematological parameters were without deviations from the standard. On the day of the admission, a sample of cerebrospinal fluid was taken from the patient for examination—a picture of monocytic pleocytosis was present in the cerebrospinal fluid, the diagnosis of serous meningoencephalitis was confirmed in the context of the clinical condition. Anti-oedema therapy was administered to the patient—corticoids and osmotic diuretics—and a strict rest regime was indicated. Cerebrospinal fluid was sent for serological and molecular genetic testing. Herpes infection and enterovirus infections were eliminated by PCR. Tickborne meningoencephalitis and Lyme borreliosis were also serologically excluded. After excluding other probable causes, the West Nile virus was suspected—cerebrospinal fluid, serum and urine were sent to the NRL for arboviruses for further analysis. The patient's health gradually improved during hospitalization, and anti-oedema doses decreased. As a complication of the corticosteroid therapy, the patient developed deep vein thrombosis of the right lower limb; given this result, anti-coagulant therapy was indicated. The complete serological results of the NRL examination were not absolutely unambiguous; admitted as a possible WNV pathogen, but Usutu virus infection was considered more likely. There is no causal treatment for any of these diseases, only symptomatic treatment is available; the patient was discharged from the hospital in good clinical condition on the 12th day of the hospitalization. At the follow-up visit on day 10 after discharge, the patient was completely free of symptoms; the neurological finding was negative.

**Epidemiological background**: Due to the development of symptoms soon after arrival in Turkey, the patient probably developed the disease before departure. The patient has a permanent residence in South Moravia and has reported mosquito biting.

### *2.2. Diagnostic Summary*

Diagnostic outcomes are summarized in Table 3. The diagnosis of our patients was based on a case definition published by European Union [EU]. RealStar® WNV RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany) assay was used for PCR and positive samples were sequenced [15]. Patient sera were tested for anti-WNV IgG and IgM with three commercial assays: Anti-West Nile virus IIFT IgG and IgM, Anti-West Nile virus ELISA IgG and IgM (both Euroimmun, Lübeck, Germany), ELISA West Nile Virus IgG and IgM capture Dx SelectTM (Focus Diagnostics, Cypress, CA, USA) and for anti-TBEV IgG and IgM with ELISA Viditest anti-TBEV IgG, IgG avidity and IgM (Vidia, Vestec, Czech Republic). VNT for WNV, USUV and TBEV according to a previously published internal protocol were used to confirm serological results [16,17].

In three patients both PCR and serology were positive, while one was not tested for PCR and one had positive antibodies but negative PCR. Only one of the WNV-positive samples (Patient 2) was confirmed to be a WNV-lineage 2 virus (by sequencing) and revealed a WNV-2 strain almost identical to WNV-2, which was detected in local mosquitoes [3]. Other samples could not be sequenced due to a low amount of viral RNA in samples. Therefore, USUV identification was not confirmed by sequencing or PCR. Positive serology was confirmed by a virus neutralization test (VNT) for WNV in all 5 patients; therefore, they met the laboratory criteria for a confirmed case. Subsequently, established VNT for USUV detected anti-USUV antibodies in 4 of the 5 previously diagnosed WNV patients. Three patients had anti-USUV VNT titers significantly lower than anti-WNV, presumably corresponding to cross-reactivity. The 5th patient had anti-USUV VNT titer 16 times higher

than the anti-WNV, implying she was most likely to have a USUV infection, although her results originally matched the EU definition for WNV. Regarding serology, cross-reactivity was detected in the ELISA, while no patient had a equivocal positive anti-TBEV VNT.
