West Nile disease (WND)
Virology and Pathogenesis
West Nile virus (WNV) is a mosquito-borne virus belonging to the genus Flavivirus in the Flaviviridae family. The viral genome is approximately 11,000 nucleotides in length coding for three structural (C, capsid protein; prM, the membrane precursor protein that is proteolytically cleaved by a cellular protease to form the M protein in mature virions; and E, envelope protein) and seven nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). The E glycoprotein is the most immunologically important protein.
Serologically, West Nile virus is a member of the Japanese encephalitis serocomplex, which includes antigenic related viruses as Murray valley encephalitis (MVE), St. Louis encephalitis (SLE), Kunjin (KUN), Usutu (USU), Koutango (KOU), Cacipacore (CPC), Alfuy (ALF) and Yaounde (YAO).
Different genetic lineages have been identified worldwide but the strains responsible for serious epidemics are attributable to Lineage 1 and Lineage 2. Phylogenetic analyses revealed that all European WNV lineage 1 and 2 strains are derived from a limited number of independent introductions, most likely from Africa, followed by local spread and evolution. Other lineages have been identified but not associated so far with human or animal diseases.
WNV is rapidly inactivated in the environment outside hosts. Low temperatures preserve infectivity, with stability being greatest below -60°C. It is inactivated by heat (50 to 60°C for at least 30 minutes), ultraviolet light, and gamma irradiation (Burke and Monath, 2001). The virus is also susceptible to disinfectants such as 3 to 8% formaldehyde, 2% glutaraldehyde, 2 to 3% hydrogen peroxide, 500 to 5,000 ppm available chlorine, alcohol, 1% iodine, and phenol iodophors.
Mosquito salivary components introduced at the site of infection in vertebrates modulate initial infection of target cells such as keratinocytes, Langerhans cell and skin-resident dendritic cells (Lim et al., 2011). The infected cells migrate to draining lymph nodes generating a viremia responsible for the infection of visceral organs and potentially to the central nervous system. How Flavivirus neuroinvasion occurs is still poorly understood. Postulated mechanisms include the direct viral crossing of the blood-brain barrier due to cytokine-mediated increased vascular permeability; the passage through the endothelium of the blood-brain barrier; a Trojan horse mechanism in which infected tissue macrophages are trafficked across the blood-brain barrier; and the retrograde axonal transport of the virus to the central nervous system via infection of olfactory or peripheral neurons (Cho and Diamond, 2012).
WNV presence and distribution
The West Nile virus (WNV) is among the most widespread mosquito-borne flavivirus in the world (Weissenböck et al., 2010). WNV was originally isolated from Uganda in 1937; afterwards epidemic outbreaks have been reported in Asia, Europe, Australia; and in August 1999 was introduced into the United States causing deaths in wild and zoo birds, horses, and humans in the New York City area. West Nile virus introduction and circulation have been demonstrated on multiple occasions in Southern Europe and Mediterranean basin since 1960s when seropositive animals or virus isolates were discovered in France, Portugal, and Cyprus (Filipe et al., 1969; Joubert et al., 1970), with WNV activity having dramatically increased over the last 10 years and spread to eastern territories without previous WNV records. If migratory birds have been associated to the introduction of viral strains from endemic areas (Calistri et al., 2010) the mechanism of virus persistence in animal hosts in Europe leading to endemization of the disease is still unknown. The circulation of WNV in Europe may occur silently for several months, or even years, before a spill over event occurs and viral circulation becoming evident. In Europe, WNV has mainly been reported in central and south-eastern Europe, regions in which WNV infections and virulence have recently increased, and the implicated viruses have spread to new areas, including Bulgaria and Greece in 2010, Albania and Macedonia in 2011, and Croatia, Serbia, and Kosovo in 2012, Germany in 2018.
Epidemiological situation in Italy
Following the first limited evidence of viral circulation in Tuscany in 1998, the Italian Ministry of Health issued a national veterinary surveillance plan for WNV monitoring areas at risk for WNV introduction and circulation. The surveillance system did not detect any relevant circulation of WNV until 2008, when the virus was identified in mosquitoes, birds, horses and humans in the area surrounding the Po river delta (Savini et al., 2008). Since then, WNV epidemics have been registered every year caused by genetically divergent isolates (Savini et al., 2012; Monaco et al., 2015) and, to date, 15 out of 20 Italian regions are considered endemic (Go to Italian Epidemiological reports).
Surveillance activities have been refined every year according to the epidemiology of the disease in the country thus leading to the adoption of a national plan integrating human, animal and entomological surveillance (One Health Surveillance) (Italian Ministry of Health, 2019).
In the Italian ecosystem, peak transmission of WNV to humans in Italy typically occurs between July and September, coinciding with the summer season when mosquitoes are most active and temperatures are highest. The mosquitoes cease their activity during the colder months, but it has been demonstrated that the virus is able to survive during this period in the infected mosquitoes, which overwinter indoors (Nasci et al., 2001).
To early detect WNV circulation and therefore to reduce the risk of transmission to humans by triggering both vector-control and SoHO safety measures, wild birds, corvids (Eurasian jay, Carrion crow and Magpie), poultry, horses, and mosquitoes are sampled according to a risk-based ranking of the Italian provinces.
Routes of transmission
WNV is maintained in nature by enzootic cycle between adult ornithophilic mosquitoes and several bird species. Birds are the WNV reservoir and play an essential role for the amplification and spread of virus in news areas. WNV vectors in Europe belonging to the Culex, Aedes,and Coquillettidia genera, which feeds on a wide variety of vertebrate host species. Mosquitoes became infected biting on a viraemic bird. After blood meal, WNV is able to spread through the gut wall into the haemolymph, replicates in most of the internal tissues and reaches the salivary glands. The period from the assumption of the virus until its localization in the salivary glands of the vector is named "extrinsic incubation period". Furthemore, in a few birds species susceptible to infection, a direct oral route transmission of the virus has been described (Komar et al., 2003). It is likely that practices such as assembly, feeding of nestlings, cannibalism, predation and necrophagy may allow the viral spread. In vector mosquitoes is vertical transmission is possible (Reisen et al., 2006) but the epidemiological impact considered negligible.
When ecological and climatic conditions favour substantial viral amplification within the birds-vector transmission cycle, increasing numbers of infected mosquitoes present a human/horse infection risk (epidemic cycle) trasmission.
West Nile virus is most commonly transmitted to humans by mosquitoes even though additional routes of human to human transmission have also been documented as blood transfusions (Pealer et al., 2002) , organ transplants (Nett et al., 2012), exposure in a laboratory setting (Campbell et al., 2002) or the transmission from the mother to baby during pregnancy, delivery, or breastfeeding (Petersen and Hayes, 2008). It is important to note that these methods of transmission represent a very small proportion of cases thus sufficient to evoke only a sporadic occurrence of the disease. Humans are dead-end hosts since are not able to infect mosquitoes during the viremic phase of the infection. Thus, the above mentioned routes of direct transmission represent the main risk of infection dissemination among community. To minimize the risk, the screening of blood and organs for transplantation in areas with WNV circulation is a common measure to prevent any inter-human spread. Laboratory acquired infections have also been reported.
WNV is transmitted by different genera and species of mosquitoes. The main vectors are some of the species of ornithophilic mosquitoes belonging to the genus Culex, which is always closely associated with the transmission of WNV during outbreaks. Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during colder months (Mancini et al., 2017). The mosquitoes cease their activity during the colder months, but it has been demonstrated that the virus is able to survive during this period in the infected mosquitoes, which overwinter indoors.
More information about WNV vector distribution are available at the links below:
• EFSA Panel on Animal Health and Welfare, 2017, West Nile Virus
• Mosquito factsheets
Host species susceptible to WNV infection
West Nile Virus has an extremely broad host range. Many species of birds act as primary hosts for WNV, though its vertebrate host range includes also species of mammals, amphibians, and reptiles (McLean et al., 2002). Not all infected hosts transmit the virus, but only those in which the virus replicates efficiently enough to reach viremias sufficiently high to infect mosquitoes through blood feeding. Birds are the main vertebrate hosts of the WNV. It is generally acknowledged that Passeriformes (especially Corvidae, Fringillidae, and Passeridae families), Charadriiformes (Laridae) and Strigiformes are considered highly competent hosts, although differences in viremic levels vary depending on the species and the viral strain.
Horses and humans are considered dead end hosts of the virus and do not contribute to the transmission cycle as they develop a low and transitory viremia not considered able to infect competent mosquito species.
WNV has been also associated with sporadic disease in small numbers of other species, including squirrels, chipmunks, bats, dogs, cats, white-tailed deer, reindeer, sheep, alpacas, dromedary camels, alligators and harbour seals during intense periods of local viral activity. Some species of mammals including squirrels (Sciurus sp.), eastern chipmunks (Tamias striatus) and eastern cottontail rabbits (Sylvilagus floridanus) may be capable of transmitting WNV to mosquitoes, although their importance as reservoir hosts is still uncertain.
Among reptiles, clinical signs were mainly reported during outbreaks in alligators and crocodile monitor (Varanus salvadori) lizard. Amphibians including lake frogs (Rana ridibunda) and North American bullfrogs (Rana catesbeiana) can also be infected with WNV. As with mammals, their importance as reservoir hosts is still uncertain.
- WNV presence and distribution
West Nile virus causes disease in humans, horses, and several species of birds. Most infected individuals show few signs of illness, but some develop severe neurological illness which can be fatal.
Most species of birds can become infected with WNV. Incubation period usually is 3-4 days and the clinical outcome of infection is variable. Some species appear resistant while others suffer fatal neurologic disease. Commonly symptoms observed are:
• ruffled feathers,
• weight loss ataxia,
• handling movements,
• stiff neck,
• motor incoordination.
Death usually occurs 24 hours after the onset of nervous symptoms.
The incubation period for equine WN encephalitis following mosquito transmission is estimated to be 3-15 days. A fleeting viraemia of low virus titre precedes clinical onset (Bunning et al., 2002). WN viral encephalitis occurs in only a small per cent of infected horses; the majority of infected horses do not display clinical signs (Ostlund et al., 2000). The disease in horses is frequently characterised by mild to severe ataxia. Additionally, horses may exhibit weakness, muscle fasciculation and cranial nerve deficits (Cantile et al., 2000; Ostlund et al., 2000; Snook et al., 2001). Fever is an inconsistently recognised feature. The clinical signs can resolve with healing in 5-15 days or progress rapidly with death of the subjects. Treatment is supportive and signs may resolve or progress to terminal recumbency. The mortality rate is approximately one in three clinically affected unvaccinated horses.
Most people infected with West Nile virus do not develop any symptoms. The incubation period for clinical illness generally ranges from 2 to 14 days, but prolonged incubation periods of up to 21 days have been observed among immunocompromised patients. West Nile fever can range from a mild infirmity lasting a few days to a debilitating illness lasting weeks to months. Symptoms are of sudden onset and often include headache, malaise, fever, myalgia, chills, vomiting, rash, fatigue, and eye pain (Zhou et al., 2010). Less than 1% of people infected via mosquito bite develop West Nile Neuroinvasive Disease Meningitis characterized by clinical signs of meningeal inflammation, including nuchal rigidity or photophobia associated with encephalitis characterized by depressed or altered level of consciousness, lethargy or acute flaccid paralysis (Sejvar et al., 2008). All ages are affected, although very strong predilection is shown with advancing age. Case fatality rates among patients with neuroinvasive disease generally approximate 10%. Advanced age is the most important risk factor for death, ranging from 0.8% to 17% in those aged at least 70 years (Lindsey et al., 2010). Treatment is supportive and illness duration varies from weeks to months with possible long-term functional and cognitive difficulties.
Anatomo pathological lesions
In birds the most important lesions are characterized by:
• meningoencephalitis with a marked involvement of the Purkinje cells of the cerebellum, (Monaco et al., 2015)
• liver and kidney involvement.
There are no macroscopic lesions affecting the organs, the lesions are visible only at the microscopic level and are exclusively affecting the central nervous system.
Anatomo-pathological lesions in humans are limited to the presence of necrotic foci with infiltration of polymorphonuclear leukocytes and macrophages affecting the central nervous system, liver and heart.
Due to the occurrence of inapparent WNV infections, diagnostic criteria must include a combination of clinical evaluation and laboratory tests. Moreover, diagnostic tests are influenced by the great variability of the West Nile virus genome, the cross-reactivity with other Flaviviruses, the transient viraemia and the low viral load developed during the infection. These elements hamper the diagnostic pathway, which must be performed by specialized laboratories and reference centers.
In Italy, all the samples collected within of veterinary surveillance are tested by the network of the Istituti Zooprofilattici Sperimentali (IIZZSS) located throughout Italy and positive samples have to be confirmed by the National Reference Center for the Foreign Diseases of Animals (CESME) held by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM).
The available and most used tests for the direct and indirect diagnosis of WNV infection are (OIE Terrestrial Manual Chapter 2.1.24 2018):
• Molecular biology techniques (RT-PCR and real time PCR);
• Viral isolation,
• IgM ELISA,
• IgG ELISA,
• Microtitre seroneutralization,
• Plaque reduction neutralization test.
In the last years, the enzyme immunoassays (ELISA) have been widely used due of their great specificity and sensitivity, as well as rapid execution. However, cross-reactivity between Flaviviruses belonging to the same serocomplex, as for West Nile and Usutu viruses, requires to confirm ELISA positive results by using the seroneutralization test. Within the national veterinary surveillance activities the diagnostic to define a suspected and confirmed case of WNV infection are defined in "Integrated national plan for prevention, surveillance and response to West Nile and Usutu viruses - 2019"".
- Clinical signs
Samples to collect
• Whole blood or serum,
• EDTA blood,
• Cerebrospinal fluid (CSF).
• Brain, spinal cord, cerebrospinal fluid CSF,
• Whole blood or serum,
• EDTA blood,
• Oral and cloacal swabs.
Prevention and control
There is no specific anti-viral treatment for the disease, and prevention can be achieved by minimizing the exposure to the vector or, in equines, through vaccination. Vaccination of horses is recommended in endemic areas to prevent the development of clinical signs in the infected animals. To date, three vaccines have obtained the marketing authorization in EU member countries for horses:
• an inactivated vaccine, produced from the VM-2 strain (Equip® WNV, Zoetis, Belgium previously Duvaxyn® WNV, Pfizer, US),
• the recombinant canarypox virus vCP2017 strain, that expresses the WNV prM/prE genes (Recombitek equine WNV vaccine, Merial)
• and the inactivated chimaeric flavivirus strain of Yellow fever virus presenting the genes for the structural proteins E and prM of WNV (Equilis® West Nile, Intervet International BV, Netherlands)
(EMEA, 2008, 2011 and 2013).
No vaccines are currently available for human use.
Control of substances of human origin (SoHO)
In affected areas, all measures are applied to limit the risk of direct transmission of human to humans through substances of human origin (SoHO) in Italy, safety of donation and transplantation procedure is ensure by the systematic collection of epidemiological information on WNV in Italy and in the world implemented by National Italian Blood Centre and National Italian Transplant Centre.
In endemic areas the main strategy to reduce human risk infection is based on vectors control (Bellini et al., 2014).
and to personal protective strategies to limit mosquito bites. Preventive activities against vectors require an integrated approach or Integrated Vector Management (IVM). The World Health Organization (WHO) defines IVM as "a rational decision-making process for the optimal use of resources for vector control". IVM goal is to find the best cost-benefit combination of all the control methods available respecting the environment to reduce the density of vectors and/or vector-human contacts so that they are not a danger to public health (WHO).
Currently, vector control plans include search and removing larval sites and larvicide treatments. The most common larval sites are marshes, open ducts, perennial basins, rice fields, cisterns, purifiers etc. Larval mosquitoes control is intensified and associated with an extraordinary treatment adulticides if a high risk of transmission to humans is found (Italian Ministry of Health, 2019).
The best protection from mosquito-borne diseases is preventing mosquito bites indoors and outdoors, especially from sunrise to sunset when mosquitoes are most active. Such measures include: use mosquito repellent, wearing long-sleeved shirts and long trousers using mosquito nets.
Information about vector surveillance and control strategies are provided at the links below:
• CDC - Integrated Mosquito Management;
• Guidelines for mosquito surveillance;
• ECDC - Prevention and control measures for West Nile fever;
• EFSA - Malattie trasmesse da vettori.
• Indications of Blood National Center
• Indications of National Italian Transplant Centre
• Web page for Italian Ministry of Health dedicated to West Nile virus
• Web page for ECDC dedicated to West Nile fever
• EFSA's documents dedicated to West Nile virus:
• West Nile fever
• Vector-borne diseases
• OIE's web pages dedicated to West Nile virus:
• Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2019
• The OIE Terrestrial Animal Health Code (the Terrestrial Code)