West Nile virus (WNV) infection is a cause of a prominent arboviral infection whose agent is a member of Japanese encephalitis serologic complex that includes a lot of human pathogens such as St Murray encephalitis, Japanese encephalitis, yellow fever, and dengue viruses. WNV was isolated in 1937 from a woman with febrile disease in the West Nile region of Uganda as one of the first arboviruses to be identified. The virus has a single-stranded positive-sense RNA genome and taxonomically belongs to the genus Flavivirus in the family Flaviviridae. Phylogenetic analysis of WNV indicated that it has two well-described genetic lineages, WNV Lineage 1 distributed all over the world and Lineage 2 which is only seen in sub-Saharan Africa. Its natural transmission cycle consists of mosquito members of the genus Culex and ver-tebrate hosts including some birds that are considered as amplifying and disseminator reservoirs[4,5].
Furthermore, birds and horses are the host species that are considered to be involved directly in the enzootic cycle of the virus. The initiation of the enzootic cycle of the virus in infected mosquitoes feeding on non-immune birds paves the way for the development of high viremia in some bird species, allowing for the transmission of the virus to uninfected mosquitoes to continue the cycle. Even though more than 100 North American bird species get infected by WNV, high viremia is not observed in all of them. While mild or asymptomatic illnesses with WNV in some avian species are common, the virus also causes infection in crows from the Corvidae family, resulting in a large epizootic disease with a high die-off which may be accepted as an important sentinel indicator of subsequent human WNV infections.
The symptomatic infection develops mostly in mammals, particularly in humans and Equidae, which are considered accidental hosts. Meanwhile, there is no significant contribution of either species on the viral amplification cycle because the viremia phase is short and low in titer.
The first route for the transmission of WNV to humans is mosquitoes of the genus Culex. Contaminated blood transfusion and organ transplantation are also the other ways of transmission[7,8,9]. Furthermore, there is evidenced transmission to children from WNV infected mothers via delivery or breastfeeding. In terms of clinical perspective, the incubation period is 3–14 days. Most WNV infections in humans are asymptomatic or have mild influenza-like symptoms. Acute systemic febrile illness occurs in 20–30% of people contracting WNV. On the other hand, it may be life-threatening in 1% of cases, depending on the developing neurological disorders like meningitis or meningoencephalitis. Particularly, elderly and immuno-compromised patients are always thought to be at risk of WNV infection due to this neuroinvasive form which may lead to death. From the past years to the present, there are many reports in Turkey on investigating the serological status of WNV in mammalian species including humans. Although the presence of WNV in humans in Turkey is reported from the 1970s with both serological and molecular studies, there are no serological studies related to WNV in humans who live in the provinces of the Black sea Region in Turkey except for Zonguldak, another province in this region.
The current study aimed to serologically investigate the presence of WNV in humans living in Samsun, the biggest city in the Blacksea region of Turkey, and it aims to obtain epidemiological data that will contribute to the implementation of public health policies to control and prevent potentially other life-threatening arboviral infections.
MATERIAL & METHODS
This study was planned in Samsun, the biggest city on the northern coast of Turkey with a population of over one million. Sampling was conducted at a tertiary level in the Samsun Training and Research Hospital, which provides healthcare services to 750,000–800,000 people every year. Blood samples of randomly selected 416 patients who came to the various clinics of the hospital for medical examination and surgery were collected between June and August in 2020 to screen both IgM and IgG antibodies against WNV. Although most of the sampling included patients from Samsun downtown and its boroughs, there were also other patients from 20 different provinces who came for summer break. After that, the laboratory works were done at Ondokuz Mayis University. 45.45% of the patients were female (n=189), and the remaining 227 (55.55%) were male. The patients were grouped into four age categories as shown in Table 1, consisting of 0–20, 21–40, 41–60, and >61 years.
To detect the presence of both IgG and IgM antibodies to WNV in serum samples, commercial Anti-West Nile virus ELISA kits (Euroimmun AG, Germany) were employed according to the manufacturer’s instructions. After the plates were read on a 450 nm filter using an ELISA reader (Rayto, China), the results were evaluated according to optical density (OD) values. To confirm the validity of tests, the results were calculated with the ratio (R) using the formula given below.
The results were interpreted as negative if R< 0.8, borderline if R ≥ 08 to a < 1, and positive if R ≥1.1.
In addition, WNV-IgG positive serum samples were subjected to a standard microneutralization test (SMNT) to confirm the presence of WNV. Briefly, positive serum samples were inactivated at 56°C for 30 min. Two-fold dilutions of serum samples were made up between 1:5 to 1:1250 using EMEM and were plated in quadruplicate in 96-well plates using 50 µl of each serum dilution. 50 µl of 100 TCID50 of WNV NY99 strain was added to the corresponding wells and incubated at 37°C for 1 h. Then, 50 µl of 5.0 × 105 Vero cells were added to each well, and the plates were reincubated at 37°C for 96 h in a humidified incubator with 5% CO2 until the appearance of an easy detectable cytopathic effect (CPE). The dilution of serum reducing CPE in 50% of infected cell cultures was determined.
The viral RNA was extracted using the GeneJet RNA purification kit (Thermo UK). Real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) was used as previously described. Briefly, PCR was performed using the iTaq Universal Probes One-Step Kit as directed by the manufacturer on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). Primers and probes of the final concentration were 320nM and 160nM, respectively. The reaction volume was 25 mL, which consisted of 12,5 µL of 2X iTaq universal probes reaction mix, 0.8µl of the forward (5’- TCAGCGATCTCTCCACCAAAG-3’), and the reverse (5’-GGGTCAGCACGTTTGTCA TTG-3’) primers, 0.4 µl of probe (5’- FAM- TGCCCGACCATGGGAGA- AGCTC-TAMRA-3’), 0.5 µl of the enzyme, 5 µl of template RNA, and 5 µl of RNase free water. PCR was subjected to cycling conditions, including 50°C for 10 min (reverse transcription), 1 cycle at 95°C for 3 min, followed by 35 cycles at 95°C for 10s and 55°C for the 20s, and 1 cycle 10 min at 72°C.
This study was conducted with the approval of the Ethical Committee of Ondokuz Mayis University for Clinical trials. (No.: 08/1523-163, date: 15/05/2018).
RESULTS & DISCUSSION
We used pooling methods to screen both IgM and IgG antibodies against WNV. A total of 42 pools were created from 416 serum samples to detect IgM antibodies [Table 2]. Each of the 41 pools included 10 serum samples, whereas only one pool was composed of six samples. Four (9.52%) of the 42 pools were detected as positive. Following this, seropositive pools were tested individually to determine the positive samples. There were two positive samples in one pool, whereas one positive serum sample was detected in each of the remaining three positive pools. The overall IgM seropositivity rate of WNV was found as 0.96% (4/416). All IgM positive individuals were from Samsun province. WNV-IgM antibodies were detected in none of the WNV-IgG positive individuals. Furthermore, all IgM positive samples were also tested using real-time RT-PCR in terms of WNV-RNA and were determined to be negative.
A total of 83 pools were made up of 416 serum samples for the screening of IgG antibodies. While 82 of these pools consisted of 5 serum samples, only 1 pool had 6 serum samples. All pools were tested using the Anti-West Nile virus ELISA IgG kits. Three (3.61%) of the 83 pools were interpreted as positive. Then, three seropositive pools were tested individually to determine the positive samples. Only one positive serum sample was detected in each positive pool, and the overall IgG seropositivity rate of WNV was found as 0.72% (3/416). Of the three positive samples, one patient was from Samsun, whereas the remaining two were from Tokat and Ordu, known as adjacent provinces. All WNV-IgG positive samples with ELISA were confirmed by detecting high antibody titers (> 160) using SMNT.
West Nile virus is the leading cause of mosquito-borne zoonosis in many parts of the world. Even though its original prevalence was in Africa, Australia, some parts of Europe, and West Asia, it widely spread from Canada to Venezuela following its introduction to the USA in 1999. Since the first isolation in 1937, many WNV outbreaks influencing humans have occurred in Israel, Greece, Romania, Russia, and USA. WNV cases occur as spatio-temporal; so, the majority of epidemics occur in the Mediterranean Basin and Central Europe between May and October, these two months are the time for the migration of birds when the number of mosquitoes is intensive. In 2019 and 2020, a total of 779 human WNV cases, 88 of which resulting in death, have been reported in the European Union (EU) member states. As well as EU neighboring countries involving Turkey; and 63.8% (497/779) of these cases were reported from Greece, Bulgaria, Hungary, Romania, and Cyprus which surround Turkey[13,14]. By its location Turkey is always at risk of having WNV cases due to its closeness to these countries, having mosquitoes in abundance, and being in the migration route of birds.
WNV-RNA is detectable in peripheral blood from 2-3 to 14–18 days. Thus, it can also be diagnosed by using molecular methods in serum, plasma, and cerebrospinal fluid. Sometimes, there can be disadvantages due to the exceptional circumstances accounting for negative results depending on the low level and short viremia. However, viral RNA detection for the diagnosis of WNV infection is still rapid and sensitive method, not only for human samples but also for animal tissues and nonhuman samples such as mosquitoes. Nowadays, serological methods are a commonly applied approach for the routine diagnosis of WNV infection[14,15]. ELISA is at the forefront among them for being easy to use in the laboratory setting, having automated protocols, and being reproducible.
In the current study, we investigated 416 patients attending various clinics of the tertiary level hospital for medical examination in terms of the IgM and IgG antibody prevalence of WNV by using ELISA. The presence of anti-WNV IgM was determined in 4 (0.96%) of the 416 participants. None of those had symptoms regarding WNV infection; so, they were asymptomatic individuals. Considering that 80% of the WNV infections were asymptomatic, the detection of IgM could be interpreted in terms of two perspectives: (i) either IgMs could indicate active WNV infection due to being determined in serum as from day 4, or (ii) WNV IgMs could also reflect the past infection and recovery because of being persisted in serum for months and even years after infection[16,17,18]. Furthermore, it could be possible to detect the viral genomes in serum or plasma samples during this period because of the possibility to identify WNV-RNA in the blood from 2–3 to 13–14 days post infection. Therefore, the presence of WNV-RNA was investigated in WNV-IgM positive serum samples with real-time PCR. None of them were positive in terms of WNV-RNA. This may depend on two causes (i) the level of RNA had peaked before symptoms appeared, then rapidly declined over several days as antibody production began; so, it had been below the detectable level when IgM was easily detectable[18,19] and, (ii) it indicated the past WNV infection in participants who were IgM positives due to the long-lasting IgM response.
Flaviviruses have a close antigenic relationship leading to cross-reaction among them. Therefore, the results should be confirmed by further serological tests, in particular, the plaque reduction neutralization test (PRNT) that is commonly used. The SMNT test could be considered as one of the confirmatory tests in European countries where the cross-reactions between WNV and other flavivirus infecting humans are uncommon. In the current study, 3 of the 416 (0.72%) participants had IgG antibodies against WNV, indicating the past infection. Furthermore, the confirmation of those in terms of whether all were specific to WNV was conducted with SMNT. All had over 1/160 antibody titers which were interpreted as robust evidence of WNV circulation.
Many studies on the presence of WNV in humans have been conducted in Turkey since it was first reported in 1970. The seropositivity percentage of WNV changed in these studies between 0.9% and 57% depending on the method used, according to the data outlined previously. However, there is no study on the prevalence of the stated virus in residents of Samsun and neighboring provinces, whereas there are many WNV studies in animal and avian species. Although data from the current study were lower than previous studies reported in Turkey[7,8,23], it was the first notification revealing the presence of WNV in humans who were living in Samsun province and its neighborhood.
In conclusion, the current study presents for the first time a dynamic WNV circulation in individuals living in the Samsun area, the biggest city on the northern coast of Turkey. Considering that WNV outbreaks are also observed in neighborhood countries like Greece year by year, it is likely that Turkey is at risk of a WNV outbreak depending on its geographical location. Therefore, (i) the preparation of an integrated national surveillance plan, also including the periodical monitoring of WNV activity, is recommended, (ii) even though 80% cases are asymptomatic, WNV should be taken by clinicians into consideration in the differential diagnosis of unknown neuroinvasive syndromes like meningitis and meningoencephalitis and febrile diseases, especially in the summer period, and (iii) horses and birds should be monitored in rural areas as sentinel animal species.
We anticipate that these outcomes will provide a basis for further studies regarding the continuous monitoring and surveillance of West Nile virus (WNV) infection as we concluded that WNV is in circulation in some northern provinces of Turkey, which means people who live in those areas are under the risk of infection; thus, more research focusing on risk factors and epidemiology of the disease is in need for endemic countries like Turkey.
Conflict of interest:
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