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Note. Adapted from Habarugira et al., (2025), Tissue, tropism, pathology, and pathogenesos of West Nile virus infection in saltwater crocodile (Crocodylus porosus), licensed under CC BY 4.0. https://creativecommons.org/licenses/by/4.0/
Alligator mississippiensis
Crocodylus porosus
Crocodylus niloticus
Crocodylus moreletii
(Habarugira et al., 2020; Machain-Williams et al., 2013)
West Nile Virus (WNV) is a flavivirus in the Flaviviridae family, which includes over 70 viruses [5]. These are enveloped, positive-sense viruses with single-stranded RNA genomes [14]. Compared to negative-sense RNA viruses, which must first transcribe their genomes into positive-sense RNA, positive-sense RNA viruses can skip this step and act directly as mRNA, allowing for faster replication [21].
WNV has a roughly spherical, icosahedral (i.e., 20-sided) protein capsid shell that protects the virus's genetic material, and it is about 50 nm in diameter. Its pathogenesis is not completely understood; however, it is believed that WNV most often enters the skin through mosquito saliva. The virus then infects keratinocytes, which produce keratin and create the skin's protective barrier, and Langerhans cells, specialized dendritic cells that serve as immune cells in the skin. Infected immune cells carry the virus to nearby lymph nodes, where it continues to replicate. The virus can then enter the bloodstream, where it impacts various organs. Disease severity depends on how the virus spreads and the host's immune response. For example, in more severe cases, neuroinvasion can occur when the virus crosses the blood-brain barrier and infects the central nervous system [5,20].
History
The first discovery of WNV was made in 1937 in the West Nile district of Uganda, where a human patient presented with fever [17, 19]. Then, outbreaks occurred in the Mediterranean region during the 1950s, beginning in Israel and Egypt. The outbreak in Egypt, which was notably large and lasted for around three years, identified mosquitoes as the primary vectors. Another significant finding concerned outbreaks in Europe around 1996, which demonstrated a shift in the population affected from rural to urban areas. The majority of these infected individuals exhibited neurological symptoms [19].
The first detection of WNV in North America occurred in 1999, starting in New York [1,10]. During this time, horses, birds, and humans experienced the highest mortality rates. This WNV strain was very similar to the one affecting geese in Israel in 1998 [17]. However, another significant WNV strain endemic to Australia, the Kunjin strain, had a major impact in 2016, harming commercial crocodilian farming [4]. This strain of WNV has been present in Australia since the 1960s, primarily affecting humans and horses [17].
Additionally, there was an outbreak in the southeastern United States from 2001 to 2003. This primarily affected American alligators (Alligator mississippiensis) [2,16]. During this time, farmed alligators from Florida, Georgia, and Louisiana were most affected, with neurological signs being the primary clinical findings [10,15,16]. However, blood tests used to detect antibodies to determine previous exposure to WNV have been found in free-ranging alligators in Louisiana and in wild and captive alligators in Florida [4].
Clinical Signs and Progression
Note. From Habarugira et al., (2025), Tissue tropism, pathology, and pathogenesis of West Nile virus infection in saltwater crocodile (Crocodylus porosus), licensed under CC BY 4.0. https://creativecommons.org/licenses/by/4.0/
The severity of WNV varies among hosts, often as neurological symptoms or superficial skin lesions. A significant threat occurs to commercial crocodilian farming, which relies on producing and selling leather for profit [11,16]. American alligators (Alligator mississippiensis), in North America often exhibit neurological and gastrointestinal clinical signs [7], resulting in higher mortality rates [9]. Neurological signs included stargazing syndrome (i.e., extension of the neck and head upward followed by tremors and failure to right themselves), tremors, unstable and uncoordinated swimming, and opisthotonus (i.e., marked by severe spasms resulting in extreme arching of the neck, head, and spine backward) [2,14]. Gastrointestinal signs included bloating, necrohemorrhagic enteritis (i.e., inflammation, tissue death, and bleeding of the intestinal wall), anorexia, and stomatitis/mouth rot [2,5].
Skin lesions have been identified primarily in North America (e.g., alligators) and Australia, specifically in saltwater crocodiles (Crocodylus porosus). These lesions are known as lymphohistiocytic proliferative cutaneous lesions (LPCLs) or also “pix lesions” [7,16]. These lesions are very small, often around 1 to 2mm in length, and described as icepick-like indentations (Figure 1). However, these become especially visible during the tanning process on affected areas of the skin, such as the tail, mandible, and belly [9,12].
Microscopic features of WNV lesions include infiltration of inflammatory cells, such as lymphocytes and macrophages [14]. Additionally, lymphoid aggregates, which are clusters of immune cells, have been observed in various tissues, including the intestines, kidneys, liver, spleen, stomach, and pancreas (Figure 1) [7].
Transmission and Epidemiology
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Note. Designed by Freepik
WNV infection is a vector-borne disease mainly transmitted by mosquitoes and ticks [18]. Specifically, Culex mosquitoes, including Culex quinquefasciatus, Culex tarsalis, and Culex pipiens, are strongly associated with infection (Figure 2). In addition, severa birds have been shown to play a role in the mosquito transmission cycle, as they can act as primary amplifying hosts and transmit the virus to a large portion of the population (Figure 3) [1].
Additional transmission methods include direct contact. Environmental transmission can also occur through contaminated water, and farming facilities are highly vulnerable to this route. This has been due to viral shedding from the cloaca of crocodilians [1,2]. Lastly, the consumption of contaminated meats such as horsemeat can provide another method of transmission to crocodilians, as it happened in 2002 in Georgia [9].
Diagnosis
Clinical examination, such as the presence of neurological signs and skin lesions, can serve as an initial indicator of disease. Microscopic examination of these lesions, followed by immunohistochemistry (IHC) techniques, can further confirm the suspicion. This technique uses antibodies and staining to detect specific antigens in tissue samples and is typically performed post-mortem because it requires tissue collection. However, it is not limited to a post-mortem procedure [5].
WNV in crocodilians is diagnosed through quantitative reverse transcription-polymerase chain reaction (qRT-PCR), a modern molecular diagnostic method that combines reverse transcription of RNA into cDNA with real-time PCR, utilizing fluorescent dyes or probes to monitor amplification and enable highly sensitive quantification of viral load. Enzyme-linked immunosorbent assays (ELISA) are another laboratory technique used to detect antibodies associated with previous WNV infection [20,11]. Additional testing that uses biological samples to detect antibodies associated with WNV infection includes immunofluorescence assays (IFA) and microsphere immunoassays (MIA). Lateral flow assays (LFAs) are used as a faster diagnostic tool for field testing [11].
Treatment and Prevention
There is no specific vaccine or medication for treating WNV infections in crocodilians [3,20]. However, a WNV vaccine originally developed for horses by Boehringer Ingelheim Vetmedica has been used off-label to prevent fatal WNV progression in alligator farms. This approach needs further investigation, as the WNV viral genome continues to be detected in vaccinated alligators. Australia has proposed a vaccine for saltwater crocodiles (Crocodylus porosus), known as the BinJV/WNV vaccine, that uses the insect-specific Binjari virus, a flavivirus. This vaccine primarily protects against skin lesions and viral shedding associated with WNV infection [3].
Since there is no cure or direct treatment for the infection, prevention strategies are the best way to control the disease's spread. This includes managing mosquito populations in heavily affected areas. Suggested environmental friendly methods for this include botanical pesticides and essential oils (e.g., peppermint, lavender, and rosemary) [20]. Additionally, insect repellents containg DEET offer longer protection against WNV. The use of genetically modified mosquitoes has been proposed for Culex mosquitoes to reduce their ability to act as vectors for WNV; however, this approach requires further research, as it has been primarily applied to Aedes species [20]. Additionally, control over birds’ exposure to farm waste should be implemented [12].
Treatment is mainly supportive care and includes ensuring that crocodilians are continuously monitored and that appropriate temperature levels are maintained. Infected crocodilians should be quarantined, dead animals should be removed immediately, and contaminated objects should be frequently sterilized or discarded. Finally, ongoing disease surveillance should be conducted to prevent the spread of illness [12].
Further Research
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WNV has been present since its discovery in 1937 and is known to infect a wide range of susceptible hosts [17]. However, certain aspects of its infection in crocodilians require further research, including the pathogenesis of skin lesions, non-vector-borne transmission routes, WNV strain variation, vaccine efficacy across different crocodilian species from other regions, and the use of genetically modified Culex mosquitoes [1,3,16,17]. Additionally, co-infections with other pathogens warrant further research, as they can affect disease progression [4].
Readings
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