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West Nile virus: A re-emerging pathogen revisited. Cerebrospinal fluid CSF examination generally shows lymphocytic pleocytosis, but neutrophils may predominate early in the course of illness [ ]. The pathological findings are typical of a viral meningoencephalitis and include microglial nodules, perivascular chronic inflammation, and variable neuronal loss with necrosis or neuronophagia [ 3 ]. Complications are often seen in patients with WNND, and include malaise, tremor, headache, amnesia, and depression, while several ocular complications, such as optic neuritis, choriorenitis and retinal heamorrhages, have been reported as well [ 96 ].
A follow-up study in 22 WNND survivors 20 patients with encephalitis, two with meningoencephalitis in Greece, where WNV lineage 2 caused large outbreaks for three consecutive years , showed that the most common symptoms remaining after one year of infection were anorexia It is worth mentioning that during the WNV outbreak in in Greece, it was observed that although IgM and IgG antibodies were present early in the WNND cases, a delay in antibody production was observed in the non-neuroinvasive cases [ ].
During a study in Greece, a second serum sample was taken from 29 WNV infected patients 75— days after symptoms onset and a third sample was taken from eight out of the 12 patients that had a positive for IgM second sample. Is should be always kept in mind that cross-reactions in serology are common among flaviviruses [ 92 ], and caution is needed for the interpretation of serological results of flaviviral infections.
Efforts have to be focused on the development of novel diagnostic assays with increased specificity [ ]. Plaque reduction neutralization test PRNT is used for differentiation of the antibodies produced against different flaviviruses, but it is a time-consuming examination that is usually performed only in reference laboratories.
Best results are obtained when convalescent sera are tested. Moreover, some degree of cross - reaction in PRNT may cause ambiguous results. A detailed travel and vaccination history previous vaccination against Yellow fever and Japanese encephalitis viruses is essential for WNV serology results interpretation.
However, polymerase chain reaction PCR is not very helpful for the diagnosis of WNV infections due to low and short viremia in patients. Studies in animal models showed that WNV is shed for a longer time in the urine of WNV patients [ ], and recent reports showed that WNV RNA is detectable in urine at a higher rate and load and for longer time than in blood; therefore, urine might be better sample for the WNV molecular diagnostics [ 44 , 48 , ].
Virus isolation is rarely used for diagnostic purposes; it is performed in reference laboratories and it is of limited use because of the short duration of viremia and the low titer of the virus in the bloodstream of WNV-infected patients. In conclusion, the best method for laboratory diagnosis of WNV infections is the application of serological methods accompanied by confirmatory examination and combined with molecular methods performed preferably on urine samples when needed.
There is no specific treatment available for WNV infection and those with mild symptoms do not need any special care. Severe cases are provided with supportive care; in WNND cases, hospitalization is required, while critically ill patients may need special management in an intensive care unit. Encephalitis cases, in particular, should be constantly monitored for elevated intracranial pressure or seizures, while special attention should be given in cases that may need respiratory support [ 97 ].
Interferon-a and ribavirin, usually used for the treatment of viral infections, have been tried as specific WNV treatments but only in vitro , as no clinical trials have been completed [ ]. It has been hypothesized that one of the modes of action for ribavirin against WNV is the error-prone replication [ ].
Drugs that can alter the cascade of immunobiochemical events leading to neuronal death may be potentially useful high-dose corticosteroids, interferon preparations and immunoglobulin containing WNV-specific antibodies [ ]. Intravenous administration of human immunoglobulin seems to be effective for the treatment of WNV infection [ , ]; the prophylactic and therapeutic efficacy examination gave encouraging results in mice [ ], while the immediate administration to a suspected case, despite the initial negative serology test, led to quick recovery of a West Nile virus encephalitis case [ ].
Toll-like receptor agonists and drugs that inhibit specific cytokines, as well as interferon preparations, have shown potential therapeutic efficacy, while humanized monoclonal antibodies directed against specific viral proteins have been developed and evaluated in patients with WNV infection [ ].
Despite intensive efforts towards the development of effective WNV prevention during the last decade, no licensed human vaccine is available. The fact that WNV belongs to a certain genus, having common characteristics with viruses, such as dengue virus, Yellow fever virus, Japanese encephalitis virus, enables scientists to construct a candidate based on previous vaccine discoveries that have managed to control the spread of the abovementioned pathogens.
Up to know research has offered several vaccine candidates. It is a live attenuated vaccine which is based on the licensed Yellow fever 17D vaccine virus. The preclinical studies on hamsters, mice, monkeys and horses were successful, followed by promising results in year-populations in Phase I clinical trial [ , , ].
Phase II clinical trial showed high immunogenicity and well-tolerance in both parts of the study [ , ]; the first part included individuals aged 18—40 years old, while the second part included subjects aged 41—64 years old and people over 65 years old [ ].
Successful preclinical trials in mice and monkeys led to Phase I clinical trial that is taking place now. It has gone under two Phase I clinical trials where safety, tolerability and adequate immunogenicity were shown in groups of toyears-old and toyears-old subjects [ , ]. A Drosophila -expressed subunit vaccine candidate, named HBV also known as WNE , has been constructed based on the E protein without the transmembrane domain ; it has completed a successful Phase I clinical trial, showing adequate safety and immunogenicity levels [ ].
Some other candidates, that are now in preclinical development, include: a recombinant subunit vaccine based on soluble protein E produced in E. At the same time, many projects of inactivated vaccines, WNV protein vaccines and synthetic-peptide-based vaccines are also under evaluation [ ]. Although there is a lack of commercial interest for a human WNV vaccine [ ], a number of vaccine candidates are currently being tested that seem promising.
A comprehensive table that includes the candidates that are currently under clinical development is provided below Table 2. Even though the main mode of transmission is via infected mosquito bite, it has been shown that alternative less common modes of WNV transmission also exist: 1 through transfusion of infected blood and blood products, 2 through solid organ transplantation from an infected donor to a healthy recipient, 3 through the placenta from an infected mother to her fetus vertical transmission and 4 through occupational infection concerning mainly laboratory professionals.
Virus transmission is possible through transfusion of red blood cells, platelets and fresh-frozen plasma [ ] with the first cases being reported in [ ]. In , based on this incident, the blood-collection agencies around USA proceeded to screening of six million blood units with NAT test resulting in the removal of positive for the virus units [ ].
Although this technique is the one widely used for blood unit examination, a case of transmission despite the NAT negative result was reported in a Nebraska man that had received transfusion after surgery and presented with encephalitis after 13 days [ ], followed by a failure of NAT to detect units with a low viremia level when examined in minipools [ ]. A more targeted individual-donation NAT rather than the usual minipool examination has been suggested [ ] especially in regions with already known high WNV incidence levels, while a lot of progress has been done for more rapid and accurate assays in vitro [ ].
Meanwhile, during , a risk assessment that took place in specific metropolitan areas of the USA with high-incidence of WNV infections estimated a mean risk for WNV transmission through blood products ranging from 1. Three years after the introduction of the virus in the USA, transmission through solid organ transplantation was first reported in [ , ] followed by five additional clusters the following years [ ].
It is worth mentioning that the investigation that followed the infection of four organ recipients from an infected donor, demonstrated that the donor had been probably infected through blood transfusion [ ]. Currently, there is not any national policy that requires organ donors screening, but serious cases of neuroinvasive disease in recipients implies a need for ELISA and NAT testing of donors during transmission season [ ].
In ; a year-old WNV infected woman delivered at term a live infant with chorioretinits and severe cerebral abnormalities white matter loss, focal cerebral destruction that was positive for WNV-specific IgM and neutralizing antibodies [ ]. This was the first report of transplacental WNV transmission in humans. One case of probable WNV transmission through breast milk was reported in [ ] but since there was no confirmed case reported from that time, transmission via breast-feeding seems to be an unlikely way of non-vector-borne transmission of WNV [ 61 , , ].
Two cases of laboratory-acquired infection were reported in the USA; the most probable mode of transmission was through percutaneous inoculation [ 61 , , ] or even through exposure to aerosol [ 61 ], as shown previously in mice [ ]. Finally, in Wisconsin, during , two turkey breeders deployed a WNV clinical picture. The investigation that followed showed that the workers as well as the turkeys they were handling were WNV infected; nonetheless, the mode of transmission to these workers remains unknown [ ].
Since there is no available vaccine for human protection from WNV infection, appropriate preventive measures should be taken. People should be informed about the peak of the mosquito activity hours from dusk till dawn so that they avoid being outdoors during these hours and use insect repellents e. Protective clothing long sleeves, long pants, socks , if possible, is an additional effective measure against mosquito biting.
As for domestic protective measures, screens in windows and doors and nets especially for small children should be used, while elimination of backwater is also necessary. People should empty water from flower pots, bird baths, buckets and cans, get rid of discarded tires that can collect water and favor mosquito growth, and clean clogged rain gutters.
In addition, the use of air-conditioner or fan seems to be helpful as the cold streams prevents mosquitoes from approaching. Although it has not been proved that handling sick animals may lead to WNV transmission, CDC recommends people who handle tissues of sick animals or work in slaughtering and culling sections to wear gloves and accompanying protective clothes [ ].
Laboratory workers should take the regular precautions and try not to over-produce aerosols [ ]. Pregnant women should protect themselves from mosquito bites and should undergo diagnostic testing when necessary [ ], while the health benefits of breast-feeding and the fact that this mode of transmission seems rather not possible, there is no regulation suggesting breast-feeding stopping [ ].
Finally, laboratory testing should be implemented on blood units, especially in affected regions. Polymerase chain reaction NAT technology is generally used for blood units testing, while no policy for organ donors testing has been established so far [ ]. Table 3 summarizes the protective methods that should be followed, divided according to the modes of transmission that were previously described.
The general goal of human surveillance for WNV is to prevent severe complications and deaths linked to the virus. Specific objectives of surveillance are the disease impact assessment on public health, the monitoring of disease trend and distribution, the intervention activities evaluation and the identification of risk factors and high-risk populations [ ].
Both clinical and laboratory criteria are presented in Table 4 below. Healthcare professionals should be familiar with the case report form and the disease recording system of their country. The information that every clinician should be aware of concerning specimen sending and case reporting is provided in Table 5 that follows. Human surveillance is divided in two major categories that are presented in Table 6 accompanied by detailed explanation of each type directions.
Enhanced passive surveillance is implemented on hospitalized cases of encephalitis and WNV IgM positive patients in regions with unknown virus activity; therefore it concerns mainly primary health care providers, infectious disease physicians and neurologists. On the contrary, active surveillance should be implemented in known affected areas. Since , when the first WNV cases appeared in the USA, technological and model spatial patterns improvements offer the opportunity for risk assessment based on dynamic space-time data illustrations.
Mapping technologies are used to associate the WNV risk with habitat and virus activity characteristics. Early warning systems for WNV outbreaks can provide a basis for surveillance and targeted control interventions. The study of various diseases in relation to space is a complex science that has relied heavily on the potential of technological developments. GIS was mainly developed in the 80s, offering not only high data processing and analysis, but also accuracy [ ]. A GIS is an organized collection of computer systems engineering hardware , software systems software , spatial data and human resources to collect, record, gather data, manage and analyze information in any form relating to the geographical environment [ ].
For several years in many countries GIS and remote sensing are applied on the study of diseases and their association with environmental and socio-economic parameters. The spatial analysis is the focus of GIS systems because it includes all transformations, manipulations and methods that can be applied to geographic data in order to support decision making. Queries are the most basic analysis mode, in which the GIS system is used to respond to user needs. Examples of queries regarding issues of disease and GIS are: [ ].
The models generated by the investigator or study team should be applicable in practice, and should also be accurate, durable and effective in relation to the cost of implementation accurate, robust, cost-effective. The best evaluation is done with a control model that uses new data from the population or sample of the original model [ ].
The model evaluation is particularly important in understanding the accuracy of predictions [ ]. The effectiveness of a model can be achieved if the accuracy of model predictions is calculated [ ]. In order to develop a model of spreading, the use of two independent samples is proposed, one for setting calibration and the other for the evaluation of the model [ ]. From onwards, the virus spread to other states such as New Jersey, Connecticut, Massachusetts, and Maryland, forcing authorities to intensify their surveillance efforts by significantly developing GIS [ ].
In a recent study in Canada, GIS, mosquito population data, climate data and land use were combined to identify areas of high probability of WNV dispersion [ ]. In the state of Mississippi GIS in conjunction with data of birds positive to the virus, bioclimatic indices, vegetation and stream network used in the creation of many spatial models to identify and control the virus in spatial scale of municipalities and regions.
The system also combined data from the breeding areas of mosquitoes and dead of wild birds while in order to verify the models data from human cases were used [ ]. The use of logistic regression in conjunction with the development of GIS has increased in research relating to WNV in recent years [ , , , , ]. Logistic regression is a method of multivariate statistical analysis that uses a set of independent variables to investigate the variability of a categorical dependent variable.
Data used in the analysis are not necessary to follow a normal distribution. The prediction is based on the construction of a linear model, namely the determination of the values of the coefficients of a set of independent variables used as predictor variables.
Besides prediction, a logistic regression model enables scientists to estimate the effect of each independent variable in the value of the dependent variable. As the mean value of the continuous variable Y for a given set of values of the independent variables is estimated, with the aid of the linear regression model the probability of success p of a categorical variable value can be estimated for a set of one or more independent variables [ ].
Since GIS applications and dissemination of data systems and internet applications Web GIS have been developed in Canada in order to meet the needs of the surveillance for WNV and it is worth mentioning that the operating costs are very high [ ]. In a similar survey in Ohio, USA in early , the GIS and spatial statistical methodology was used to identify the environmental and demographic factors that have a positive effect on the risk of virus transmission [ ].
GIS combined with multivariate statistical techniques in addition to the cartography of areas with increased risk of disease allowed the determination of risk factors through a multitude of environmental and socio-economic parameters related to the ecology and epidemiology of WNV as socioeconomic conditions [ ].
Finally, in Greece although GIS is an important tool for public health risk assessment and interventions and in particular for the management and prevention of diseases like WNV, it has not yet been sufficiently evaluated. It is worth mentioning that the monitoring and control programmes launched recently aim to develop the capabilities of GIS, and to contribute with useful information and data on the spatial dimension of the WNV and its outbreaks to the decision makers to enable them to make the better decisions and take operational actions creating prevention and control techniques [ ].
This system was based on dead crow surveillance. The HIN was used by local authorities to enter data about sick or dead birds. Then, dead crows were collected for laboratory testing and the results of virus-positive birds were used in the model. The bird surveillance data and virus positive results were used to create maps which predicted viral activity [ ].
The systems used by the Department of Health in New York showed that the number of dead crows increases much faster than the number of infected birds or mosquitoes and that dead crow monitoring is much more efficient for outbreaks detection [ ].
The use of mosquito-to-crow ratio, that followed, showed that, while mosquito control decreases the virus outbreak threshold, the bird control increases it [ ]. The California State Mosquito-Borne Surveillance and Response Plan was initially developed for the surveillance and risk assessment of western equine encephalitis virus, St.
Louis encephalitis virus and other arboviruses surveillance and risk assessment and has been used since [ ]. According to this model, seven risk factors Table 7 are evaluated in a five-value rank and their average score represents the overall risk. WNV activity is categorized into normal season score: 1—2. Risk factors and thresholds for WNV risk assessment [ ].
From to , a new approach of WNV risk assessment which analyzed the risk caused by mosquito species for transmitting WNV to human was implemented in New York. This approach associated data related to abundance, infection prevalence, mosquito competence and mosquito biting behaviour. This risk assessment equation can be used as a predictive indicator to assess the relative number of future human WNV infections [ ]. The system used a geographic model based on a localized Knox test to identify the non-random space-time interaction of dead birds, as an index of an intense WNV amplification cycle using biological parameters.
This model also related surveillance data with human cases [ ]. In , Chicago competent authorities implemented the same system [ ]. Daily risk maps were open access online and used by local authorities to enhance preventive measures e. Mostashari and his colleagues used the SatScan which is a spatial scan statistic model for potential detection of infectious disease outbreaks, applied the data of dead birds reported from New York City and evaluated its effectiveness in providing an early warning of WNV activity.
All dead bird reports, mosquito traps and human cases with contact details were geocoded to a point location. Using spatial-temporal cluster analysis of dead bird reporting data, competent authorities can start early larval control activities, prioritize birds for testing and triage scarce mosquito-collection and laboratory resources [ ]. From to , a research team of Wyoming USA used a spatial analysis tool to estimate potential WNV activity using a spatially explicit degree-day model.
This model is based on GIS tool and uses temperature data, which are open access to public and user friendly software. By keeping away the areas without elevated ambient temperature, this model can predict high risk areas for WNV activity [ ]. In Colorado, a research team used epidemiological data from and for human WNV in order to quantitatively assess: 1 the degree to which spatial scale of data aggregation affected vector borne disease occurrence and 2 the extent of concordance between spatial risk patterns based on disease case counts versus disease incidence for regularly used spatial boundary units.
Maps based on spatial distribution and correlation of WNV were presented [ ]. This approach was based on the association among WNV risk, habitat, landscape, virus activity and socioeconomic parameters and used GIS analytical tools to develop WNV human risk maps [ ].
In , a research team of Montana State University USA developed a human-health risk assessment approach for WNV and associated documented health effects from WNV infection with insecticides used to control adult mosquitoes.
Human cases and exposure to adulticides were used as basis of this model. The results pointed out that human health risks from exposure to mosquito adulticides are very low and are not possible to exceed concern levels [ ].
A research team of Carroll College Montana uses another approach of WNV risk assessment model in which spatial and seasonal WNV transmission risk is estimated throughout Montana with GIS models based on the temperature threshold below which virus development will not proceed in Culex tarsalis. This model uses maximum, minimum and average daily temperature data; a degree day modeling source that produces WNV development units WDU and varying time-temperature scales throughout June, July, August, and September [ ].
WNV is locally maintained and dispersed in new areas primarily via an enzootic cycle of proliferation, which includes wild and domestic birds as reservoirs and ornithophilic mosquitoes as virus vectors. Apart from birds, the virus is transmitted to dead-end hosts as well [ 61 ]. Mosquitoes become infected by feeding on birds that carry virus particles in sufficient concentrations in their blood. Virus introduction and transmission in a non-endemic area, initiates in a wild ecosystem cycle, which involves migratory birds and then in a suburban and urban cycle, involving additionally endemic avian species.
Variations in abundance and species composition of birds species richness either periodically e. When species richness increases, a decrease in the prevalence of WNV disease is observed while the disease prevalence increases when species richness decreases [ ]. After the dramatic spread of WNV in the USA, with an initial outbreak in New York in [ ] and the upsurge of human cases in in Romania, with confirmed cases in humans [ 72 ], many studies have been conducted to determine the precise role of birds in the epidemiology and transmission dynamics of the virus.
A key conclusion that has been drawn from various experimental studies is the great diversity in the profile of viraemia among the different animal species. With an estimated limit of 10 5 plaque forming units PFU per mL of blood, above which the virus can be transmitted to a mosquito during a blood meal, different bird species can develop sufficient viraemia titers for a few days usually one to four in order to allow the transmission of the virus to the feeding mosquitoes.
These birds belong to the orders of Passeriformes corvids, sparrows, finches, etc. In contrast, species of the orders of Piciformes woodpeckers , Columbiformes doves, pigeons etc. The direct transmission of the virus between infected and healthy birds kept in the same place, without the presence of vectors, is also of importance [ ].
The fecal-oral route was considered as the most probable mode of transmission. The presence of the virus in the feces was confirmed at least in 17 out of the 24 species tested and in the oral secretions of at least 12 out of 14 species [ ]. Transmission of the virus was also achieved by feeding crows with tissues obtained from an infected dead sparrow. These data are particularly important considering that it is difficult to attribute such extensive spatial spread of the virus within a few years, as happened in North America, only to the infection of mosquitoes.
Regarding mosquitoes, a minimum period of days after infection is required to further transmit the virus to a new host. This time interval is necessary for the virus to replicate in the intestinal cells before invading other tissues and eventually reach the salivary glands, from where it can be transmitted [ ]. Therefore, it is very difficult to impute the spread of the virus at 70 km average per month for 6—8 months each year, only to the action of mosquitoes [ ].
The involvement of endemic birds and direct transmission of the virus among them constitutes a complementary mechanism of virus spread. Avian species such as corvids are feeding on corpses of animals, including infected dead birds of similar species. They are social, forming large colonies, with a daily dispersal range of up to 20 km from roost, with their area of operations overlapping with the areas of activity of birds from neighboring nests [ ]. Also, the young birds remain close to adults and assist their parents in raising their chicks.
It is therefore conceivable that birds dying from the disease may be consumed and the virus can be transmitted to birds of neighboring nests. Feeding with corpses even of infected mammals is also a likely mean of viral entrance in a flock. Lastly, the transmission can be achieved orally on chicks.
Epidemiological models support these assumptions and the apparent important role of endemic birds in the spread of the virus [ ]. WNV outbreaks mainly occur during July-September, in areas that combine the element of water with the presence of cities and a hotbed of resting for migratory birds. Migratory flocks during spring migration in April and May can carry the virus from endemic areas of Africa and introduce it in places of rest and breed in Europe.
There, a cycle of virus amplification takes place for 2—3 months between resident birds and mosquitoes, which results in a massive human exposure to the virus during the summer months. The engagement of resident birds seems necessary to justify, as mentioned above, the spread of the virus to neighboring regions. All these characteristics are evident in the outbreaks in Greece. The first outbreak occurred in Central Macedonia, an area with significant water bodies, resting area for migratory birds, with not only abundant presence of mosquitoes of the genus Culex , but also significant presence of people and great biodiversity of endemic birds.
The exposure of the endemic birds of the area was both serologically and molecularly confirmed [ 46 ]. However, the genetic similarity of the corresponding virus with isolates in Hungary and Austria in previous years [ 34 , 37 ] supports the assumption that the virus spread southwards. The autumn migration of birds from Northern Europe to wintering areas in Africa with resting stops in Greece, offers one such possible mechanism, which is supported by the detection of antibodies to WNV in young migratory birds turtle doves , born in northern Europe, on their arrival in Greek territory.
These birds could only have been exposed to the virus in the country of their origin, and this supports the hypothesis on possible entry of this strain during the past, in a similar manner. The subsequent spread of the virus, especially to Southern Europe, is combined by a correlation between areas with human cases and seroprevalence in endemic birds.
Seropositive birds have been found only in areas where outbreaks in humans have been recorded. In contrast, in survey it was found that all birds coming from areas with no reported human case were seronegative. In the region of Attica, samples obtained from a period prior to the onset of cases in humans were seronegative, indicating a possible later introduction of the virus in the region, and therefore concerns were expressed that the multiplication cycle of the virus bird-mosquito-bird is in its very beginning, expecting a significant increase in human cases, which unfortunately was confirmed for the region [ ].
The prevailing opinion on the introduction of the virus in North America involves migratory birds as carriers of the virus during their migratory journey. Indeed, this hypothesis is strengthened by the emergence of a new virus outbreak in Florida, two years after the outbreak in New York, at several hundred kilometers south.
The short duration of viremia in birds, which is sufficient for the transmission of the virus via mosquitoes, raises questions about the real possibility of birds to carry the virus to new long distant areas. Therefore, it is very likely that for an infected bird to reach a resting area, or its destination during the 48 to 64 hours after infection when viremia titer is usually sufficient for the transmission of the virus in mosquitoes fed on the blood of the viremic bird.
The arrival of migratory birds just before dawn, when mosquito activity is at peak, further supports this hypothesis. The transmission of the virus in the following days in sedentary birds of the area leads to the onset of a local enzootic cycle of WNV. The above data demonstrate the multi-factorial nature of the transmission cycle of WNV with a multitude of different climatic, environmental and ecological factors contributing to the progress of the disease spread.
At the same time, many avian species seem to be able to play an important role in the epidemiology, a fact that calls for a thorough and continuous investigation. The introduction of WNV in North America in was accompanied by high mortality in several species of birds.
More specifically, after experimental infection of American crows with the NY99 strain, birds showed signs from the fourth day after inoculation. Many signs such as depression, anorexia, decreased defecation, inability to fly, gait disorders, as well as bleeding from the mouth and the cloaca were reported.
Death occurred 24 hours later [ , ]. Similar signs occurred in other species magpies and other corvids, sparrows, finches, etc. In contrast, other species did not show any signs, and particular species belonging to classes of Piciformes woodpeckers , Columbiformes pigeons, doves etc.
Sensitive species exhibit macroscopic and histopathological lesions such as congestion of the cerebral cortex, the spleen splenomegaly was observed in some cases , heart and kidneys, dotted hemorrhages in the stomach, liver and skeletal muscle, gallbladder distension and opaque air sacs.
Histopathological examination revealed the presence of hepatocyte necrosis with cytoplasmic vacuoles, rupture of the nucleus and strong presence of mononuclear phagocytes in the tissue. The congestion of the spleen is accompanied by lymphocyte necrosis in the lymphoid follicles and increased presence of inflammatory cells, especially macrophages throughout the splenic parenchyma.
Skeletal muscle fibers were inflated with loss of striations and fragmentation of sarcomeres, while necrosis of the bone marrow has been also observed [ ]. Virus isolation from various organs is possible, both in symptomatic and asymptomatic bird species.
The virus has been isolated from the spleen, kidney, brain, heart, lungs and trachea from various species. The relatively easy isolation from feathers and skin samples is particularly interesting and enhances the probability of transmission to mosquitoes, while the isolation from the ovaries indicates a possibility for vertical transmission [ ].
In contrast to the New World, the mortality of birds in the Old World seems to be limited [ 59 , , ], although during the peak incidence in Israel — a large number of storks and geese died due to infection with lineage 1 virus strain [ ]. In —, two different strains of WNV of lineage 1 and 2 type caused fatal encephalitis in a flock of geese and various falconids, respectively, in Hungary [ 34 , ]. In , a strain belonging to lineage 2, similar to that of Hungary, resulted in death of six falconids in southeastern Austria [ 37 ].
It is therefore evident that there is a great diversity in the observed clinical signs among different bird species, as well as among similar bird species of different regions in the world, which is probably related to the history of exposure to the virus and the different immunological status of the infected birds in each case. These systems included avian-, mosquito-, and nonhuman mammal-based sampling, to facilitate prediction and prevention of human and domestic animal infections [ 40 ]. Similar arbovirus surveillance systems have sporadically been used in Europe, but there is insufficient evidence for their value as early warning indicators for WNV infection [ , , ].
Moreover, given that in Europe a variety of circulating viral strains of different virulence exists, it is important to follow a surveillance strategy, which not only would allow for early serological detection of the circulation of the virus, but would also allow for isolation and molecular characterization of the circulating strains. Such arbovirus surveillance systems may be particularly useful for better public health risk assessment, and for proper guidance regarding the implementation practices for mosquito control.
Since the beginning of WNV epidemics in Greece in the summer of and until , different surveillance systems were developed and constantly evaluated, aiming to monitor WNV circulation mainly in the Region of Central Macedonia, and also in other areas of Greece [ ]. These systems include WNV active surveillance, using domestic birds, and particularly, pigeons and chickens. A serological study was conducted in domestic pigeons Columba livia domestica , aiming to investigate their suitability as sentinels for WNV surveillance, for the assessment of the geographical spread of WNV after the epidemics of and in Greece, and also their effectiveness at alerting upon WNV enzootic circulation prior to the onset of the and epidemics in humans.
WNV circulation was detected in pigeons 1. Another study was conducted in free-ranging chickens, younger than five months old, during in Central Macedonia, with similar results [ ]. Furthermore, a serological study in captive sentinel chickens was conducted in Central Macedonia, between May—October and , in order to detect local enzootic transmission of WNV prior to the onset of WNND cases, resulting in detection and molecular characterization of the circulating strain, and early warning.
Specifically, from May , blood samples were being collected on a weekly basis from chickens which were placed in cages in the perimeter of Thessaloniki city. The samples were subjected to serological and molecular testing. In parallel to blood samplings, mosquito populations in the study area were monitored using CDC light traps [ 47 ]. The study was repeated in the greater region of Central Macedonia during [ ]. The first seropositive captive chicken was detected one month prior to the first WNND case at the same area for The virus was isolated from the blood of two sentinel chickens and the genomic region of the NS3 protein of both isolates presented highest nucleotide sequence identity For , nine WNND cases were detected in the areas which were monitored, after the detection of the first seroconverted captive chickens.
The detected strain presented highest nucleotide sequence identity The development and implementation of the aforementioned systems resulted in informing the public health authorities, in order to take timely measures to protect public health in response to the WNV epidemics of and In conclusion, it appears that determination of juvenile seroprevalence in domestic pigeons and free-ranging chickens can comprise reliable and cost-effective systems for early detection of WNV enzootic circulation.
With these systems, it became possible to study the spread of the virus in an area after the end of the epidemic. However, it should be noted that only a limited number of blood samplings was feasible to perform. On the other hand, since purchase, maintenance and care of these birds is not required performed by the bird owners , these systems are very practical and inexpensive for obtaining information on WNV circulation.
On the contrary, the application of WNV surveillance systems using captive chickens is more expensive, as bird purchase, accommodation and feeding costs are required. There are several advantages of captive chicken surveillance, compared to the previous system, because it gives the opportunity for repetitive blood sampling, aiming not only to study the geographical spread of WNV using serological methods, but also to the detection, isolation and molecular characterization of the circulating strains.
The use of captive chicken with repeated sampling is particularly useful in areas where many WNV strains of varying virulence co-circulate, and collection of this data is imperative in order to accurately determine the risk of human infection and the outbreaks of WNND cases during the epidemic period. A project for the collection of specimens of wild resident and migratory birds active since has been running in Laboratory of Microbiology and Parasitology—Department of Veterinary Medicine, University of Thessaly in the context of participation in a research programme of the EU on the surveillance of wildlife diseases FP7, WildTech.
The project was based primarily on the collection of tissue and serum samples of hunter-harvested birds collected during the hunting seasons, but also on passive surveillance using samples obtained from dead birds of unknown etiology. With the onset of the epidemic of , the project was intensified and included surveillance of both archived and the new collected samples for the presence of WNV or WNV specific antibodies.
By testing of archival samples, the exposure of sedentary wild birds Eurasian magpies to WNV was detected at least eight months before the first report of human cases [ ]. In collaboration with Hunting Federations, tissue samples and sera of hunted birds were collected from the region of Central Macedonia and the rest of Greece during the hunting seasons — and — Priority was given to search corvid samples, which have been recognized as important indicators of the presence of the virus locally, and samples of migratory birds that have been identified as carriers of WNV and meet the above requirements to transfer the virus over long distances in migration, when they are in a viremia stage.
The detection of WNV antibodies in young migratory birds on their arrival in Greece support the hypothesis described in Section 4. Horses and other equine species are susceptible to WNV infection [ ]. Unlike birds, horses as well as humans are incidental hosts of WNV [ , ].
Phylogenetic studies performed in WNV isolates obtained from equines have led to their classification into two main lineages [ 31 , 36 , , ]. Horses are characterised as dead-end hosts for the virus, since they do not develop titers of viraemia sufficient to infect mosquitoes and propagate the virus [ 31 , 36 , , , , ]. Horses, as opposed to wild birds and mosquitoes, do not perform an essential role in the spread of the virus. Direct transmission of WNV from horses to humans has been reported very rarely and only in cases handling infectious brain without special precautions during necropsy of dead horses [ , , ].
In temperate zones of the northern hemisphere, disease risk appears to be greatest during a 3-month period, between August and October, when mosquito activity peaks, whereas in subtropical regions the period of risk is longer and the outbreak pattern may not be predictable and cases may appear year-round [ , ].
Epidemiological studies during epidemics of WNF have identified important risk factors that predispose to infection of horses. Appropriate soil and climate conditions for mosquito breeding backwater, frequent rainfalls, high temperatures and the presence of domestic and wild bird species allowing ample access to blood meals, could be responsible for an increase of mosquito populations among areas where horses live.
This can lead to the easy spread of WNV, after the virus enters the ecosystem usually via migratory birds [ , , ]. Since the mids WNV outbreaks have become a major infectious disease problem in horse populations in Mediterranean countries of Western and Central Europe. In the late summer of , WNV infections were recorded for the first time in the Western Hemisphere when 20 confirmed cases in horses occurred in New York State.
WNV spread further to all the USA, Mexico, Canada and the Caribbean during the following three years with thousands of laboratory confirmed equine encephalitis cases [ , ]. The pathogenesis of WNV infection in horses is not completely understood [ , ]. The infection is initiated after the inoculation of the virus into the skin by infected mosquitoes. The virus then replicates in local tissues and in regional lymph nodes and is transported via lymphatic vessels to the blood stream.
Langerhans cells of the skin have been associated with this virus transport to the lymph nodes in WNV infections in mice. This virus replication and viremia may seed infection in extraneural tissues, increasing the virus titer in blood and perhaps preceding the invasion of the CNS [ ]. Neuroinvasion pathways for WNV are not well defined, but may involve passive diffusion across the capillary endothelium, virus replication in endothelial cells and budding of virus into the CNS parenchyma, or retro-axonal virus transport of infected neurons of the olfactory epithelium [ , ].
The low virus titer and short duration of viraemia in horses, as opposed to birds, as well as the failure to detect the antigens of WNV in vascular endothelial cells make the first two neuroinvasion pathways unlikely to occur [ , , ].
The mechanism of neurological damage is uncertain [ , ]. In a WNV infection model in hamsters it was observed that many degenerating neurons underwent apoptosis but this was not associated with inflammatory cells, so it was suggested that cellular damage was caused by WNV infection [ ]. In contrast, it was suggested that neurological damage in natural WNV infection of horses has an immunopathological component, since inflammatory changes were present in the absence of abundant detectable WNV antigens [ , ].
Equine infections either from lineage 1 or from lineage 2 WNV strains, are often subclinical, causing seroconversion in the absence of clinical signs [ 31 , 36 , , ]. In clinical cases there is a variety of signs ranging from transient neurologic deficits to fulminating fatal encephalitis.
Reported clinical signs in horses include fever, paraparesis or tetraparesis and ataxia that may be symmetric or asymmetric, recumbency and evidence of intracranial disease including vestibular or cerebellar ataxia and behavioural changes [ 31 , 36 , , , ].
These signs are not pathognomonic for WNV infection. In many clinical cases affected horses frequently show muscle fasciculation and tremors [ 31 , ]. Analysis of a large outbreak in horses revealed that the most common clinical signs were ataxia, gait disorders, muscle fasciculation, depression, and recumbency [ ].
Prognosis for infected horses is variable, with the most important contributing factors appearing to be severity of disease and vaccination status [ 36 , , ]. In contrast, as the disease becomes endemic in the region, the mortality rate among diseased horses is dramatically decreased [ ]. Sternal and mainly lateral recumbency is a strong predictor of death or euthanasia [ ]. These mortality rates include horses euthanized for welfare reasons, because of irreversible course of the disease [ , ].
In some cases the mortality rates among horses seem to be influenced by individual characteristics relating to each affected animal, such as nutritional and reproductive status or genetic factors [ , ]. Typically, in most cases of deceased or euthanized horses there are no gross lesions in the anatomical structures of the nervous system [ , , ].
However, lesions in some cases are macroscopically evident, with petechiae sparsely distributed throughout the entire rhombencephalon and extending multifocally through the entire spinal cord. These petechiae are especially prominent within the thalamus, the caudal brain stem and the ventral horns of the spinal cord [ , ].
Many changes in the internal organs and skeletal muscles can be seen as result of recumbency and other secondary complications with no diagnostic value [ , ]. The severity of lesions varies between different cases, which is not always directly proportional to the severity of clinical signs. In addition, vaccinated horses with clinical disease develop very mild and limited histopathological changes [ , ].
Histopathological lesions are detected in the lower brain stem and spinal cord, especially in thoracic and lumbar region. They are more severe and extensive in the grey matter than in white matter. Main histopathological findings are mild to moderate non-purulent encephalomyelitis accompanied by gliosis.
Histopathological lesions in extraneural tissues are extremely rare to be found in horses, unlike birds, in which can be found in many internal organs [ , ]. Particularly, histopathological lesions involve the basal nuclei, grey matter of thalamus, midbrain, lower brain stem, and ventral and lateral horns of the spinal cord. In equine WNV cases, lesions can be found in the cortex of the hemispheres and even more rarely in the cerebellar cortex [ 36 , , , , ].
The main histopathological findings are multifocal mild to severe perivascular cuffs comprised of lymphocytes and a few macrophages, with mild infiltration of lymphocytes and gliosis in the adjacent neuropil [ 36 , , , , ].
In the most severely affected tissue areas, neuronal degeneration is prominent and characterized by central chromatolysis, cytoplasmic swelling, or cell shrinkage. Small, scattered microglial foci, occasional neuronophagia, and presence of small necrotic areas comprised of macrophages, neutrophils and cellular debris may be seen.
Axonal swelling and spheroid formation is frequent [ , , ]. In addition, in some severe cases mild to severe perivascular hemorrhage with degenerative changes, local necrosis in the vascular wall and the presence of low neutrophil counts may be seen. Occasionally, mild leptomeningitis that extends as mild focal inflammation in the underlying areas of the cerebrum and cerebellum may be found [ , , ]. Application of immunohistochemical methods on tissue sections revealed remarkable features for WNV antigen distribution in the nervous system of infected horses and basic qualitative features of inflammatory infiltration [ ].
On sites of inflammatory lesions, WNV antigens are mainly localized within the grey matter and have a finely granular appearance within the cytoplasm of a few morphologically normal and degenerate neurons. WNV antigens are also present in a large number of morphologically normal nerve fibres, axonal hillocks, glial cells, and spheroids of the medulla oblongata and spinal cord.
No viral antigens have been detected within the peripheral nervous system and extraneural tissues of affected horses, which is in direct contrast to post mortem findings in birds [ ]. Perivascular and leptomeningeal inflammatory infiltration is composed almost exclusively of T-cells labelled for CD3 surface antigen.
Occasionally, the perivascular infiltration is composed purely of macrophages labelled for MAC antigen , and rare macrophages contained intracytoplasmic WNV antigens, whereas, no BLA positive lymphocytes surface antigen mainly expressed in B-cells were identified [ ]. Clinical signs of WNF in horses are not pathognomonic, so the diagnosis of infection is established by laboratory investigation.
Negative virus detection test results should thus never be regarded as evidence of absence of WNV [ 36 , , ]. Furthermore, the biochemical and cytological examination of CSF from diseased animals has no diagnostic value, because in many cases abnormal features are not found. Treatment of WNV infection in horses consists primarily of supportive care. No antiviral therapies with documented efficacy against this virus are currently available [ , ].
The supportive care is not always successful [ ]. However, recent studies have documented clinical improvement in neurologic signs when intravenous immune globulin containing specific antibodies against WNV was administered post infection.
The efficacy in animal models appears to be dose dependent; but efficacy studies in naturally occurring equine infections have not been performed [ ].