European College of Equine Internal Medicine consensus statement on equine flaviviridae infections in Europe

2.1 West Nile virus

2.2 TBEV clinical disease

Horses are prone to TBEV infection; however, they remain mostly asymptomatic. Although TBE in humans has been studied extensively, there are only a limited number of reports on TBEV infections in animals, especially in horses. Neurological signs after TBEV infection are nonspecific and can range from peracute to acute or mild subacute courses. Signs described are: reduced general condition, inappetence, hyperreactivity, ataxia, seizures, and paralysis of neck and shoulder muscles.^39-43

2.3 USUV clinical disease

Reports on clinically apparent USUV infections are scarce and few human cases are described in the literature.^{8, 44-50} Infection of humans is usually asymptomatic, but rarely serious neurological disease is observed. Pathogenicity of USUV is unclear and sometimes a case of USUV infection is wrongly identified as being caused by WNV.⁵¹ Equine USUV-related clinical disease has not been described, but horses in Poland and Croatia can be seropositive.^{52, 53} Anti-USUV antibodies were detected in 2017 in horses in Austria (unpublished data).

2.4 LIV clinical disease

Louping ill virus derives its name from an old Scottish term describing the leaping motions of encephalitic sheep. Louping ill virus causes a febrile illness in sheep, cattle, grouse and occasionally in horses and some other species, that can progress to fatal encephalitis.⁵⁴ Affected horses frequently have muscle tremors of the neck and facial area, or even generalized tremors. Paralysis, paresis of a single or all extremities, obtunded mentation, and abnormal behavior occur. Some horses are pyrexic (up to 40.5°C) and inappetent.^{55, 56}

2.5 EqHV clinical disease

Equine hepaciviraus infections are mainly subclinical with mild clinicopathologic abnormalities indicating hepatopathy.^{13, 14} Overt clinical hepatopathy in association with the infection is rarely described.^{4, 57} Equine hepacivirus infections are usually self-limiting and resolve within weeks or months. Infected horses showed a delayed seroconversion against the nonstructural protein NS3 starting from 3 to 8 weeks after infection, which often precedes viral clearance.^{3, 13, 14, 58, 59} Additionally, successful seroconversion seems to provide a nonsterilizing immunity against reinfection, leading to lower level of viremia and shorter viremia in subsequent infections.^{13, 14} Liver enzyme activities (GLDH, SDH, GGT, AST) increase around the time of viral clearance; although hepatitis lasts 1 to 4 months, peak liver enzyme activity in serum frequently remain within the reference interval.¹³ Histopathological changes are subtle, including scattered individual hepatocyte necrosis and mononuclear cell infiltrate.^{3, 13, 14}

Equine hepacivirus can also cause persistent infections. Experimental inoculation of naïve horses showed that adult horses were more likely to be able to clear the infection than young horses (8 months or younger) which implies an important role for the adaptive immune system in clearing the infection.^{2, 59} Two case reports document persistent EqHV infection with chronic hepatitis, indicating horses might develop a similar condition of chronic hepaciviral hepatitis as is observed in humans with HCV.^{4, 57} Histopathologic changes include fibrosis, hepatocyte necrosis, mononuclear inflammation, and biliary reaction.

3.1 General

The 4 genera of the Flaviviridae family have diverse mechanisms of transmission. The flaviviruses are also known as arboviruses because they are arthropod-vectored. In contrast, the pestiviruses are primarily transmitted through contact, such as fecal-oral transmission, or vertically transmitted. Method of transmission of the hepaciviruses and pegiviruses is unclear. Both are transmitted iatrogenically by blood or a blood-product transfer, but modes of natural horizontal transmission are not known.

3.2 WNV transmission

Like many other flaviviruses, WNV is mosquito vectored. It is primarily an avian virus, where birds serve as amplifying hosts (Figure 2). Over 150 species of birds can be infected with WNV.^{60, 61} More than 65 mosquito species transmit WNV, with Culex spp. being the primary vectors worldwide.^{62, 63} Mosquitoes are capable of vertical transmission and can therefore sustain the virus for a period of time in the absence of infected hosts.⁶³ While mosquitoes often have a narrow host range, those that “host-switch” or feed on both birds and mammals, such as Culex tarsalis in the western United States and Culex pipiens in the eastern United States and Europe, are responsible for the occasional transmission to horses and humans.^{63, 64} Many species of birds are susceptible to infection, some with high case fatality rate.⁶¹ There however, is variation in the reservoir/amplifying species based on geographic location.^65-67 Both changing bird and mosquito habitats and ecology can contribute to the spread of WNV into new geographic regions.

Unlike birds, horses and humans are both typically dead-end hosts. While they can develop clinical disease, they do not develop high enough viremia to transmit back to mosquitoes.^{61, 64} In humans, there is limited horizontal transmission via blood transfusion or organ transplantation, and vertical transmission through the placenta or breast milk.⁶³ These routes are not documented in horses to date.

Because WNV is arthropod-borne, there is a seasonal transmission pattern, with cases starting in the summer and continuing through the autumn, although clustering of cases within those seasons depends on the local climate.⁶⁸ In Europe, WNV outbreaks occur during the summer and autumn seasons (July-November) when Culex mosquitoes are abundant. Overwintering likely occurs through winter diapause, or hibernation, of infected mosquito females, or through transovarial transmission in mosquitoes.^{69, 70}

3.3 USUV transmission

Usutu virus ecology is remarkably similar to WNV (Figure 2), and the 2 viruses cocirculate in Europe.⁷¹ Like WNV, USUV is mosquito vectored, with birds as amplifying hosts. Culex spp., particularly C. pipiens, are the major vectors, although other genera, including Aedes, contain competent species.^{62, 71} At least 93 bird species are susceptible to infection, with blackbirds, gray owls, and house sparrows particularly susceptible to lethal infection.⁷¹ Usutu virus infects many Passeriformes that are also susceptible to WNV. Susceptible mammals, including horses and humans, are suspected to be dead end hosts, as is observed with WNV.⁷¹

3.4 TBEV transmission

In contrast to WNV and USUV, TBEV is tick-borne (Figure 3). Although more than 14 species of ticks can be infected with TBEV, Ixodes spp. are the primary vectors. Ixodes ricinus have a broad pattern of feeding on over 300 different species including mammals, birds, and reptiles. Ticks are active while temperatures exceed 5 to 7°C, which determines the seasonal transmission to mammals.⁷² In more temperate regions such as central Europe, there are 2 peaks of activity: first in May-June and then in September-October. However, in colder regions there is only 1 peak in the middle of summer. The first clinical cases are typically observed 2 to 4 weeks after the onset of tick activity.⁷²

3.5 LIV transmission

Louping ill virus is transmitted to sheep, deer, mountain hares, and grouse by feeding I. ricinus⁷³; LIV, similarly to the other TBEV subtypes, is also transmissible to offspring by goat and sheep milk and thus has a potential to be transmitted to humans.^{74, 75} Naturally acquired equine infections have occurred in enzootic areas.

3.6 EqHV transmission

Just like the closely related Hepatitis C Virus, EqHV infects hepatocytes and causes both acute and chronic infection.^{58, 59, 76} The virus can be transferred via blood, plasma, or serum,^{14, 59} including vertical transmission to the foal.^{77, 78} In contrast to other flaviviruses, hepaciviruses are not known to be insect vectored. EqHV was not detected in mosquito pools in Austria; however, this does not rule out mechanical or biological insect vectoring.⁷⁹

3.7 Geographical distribution

West Nile virus is the most widely distributed arbovirus that induces equine encephalitis (Figure 4). It was first isolated in 1937 in Uganda,⁸¹ then detected in a few European countries between 1958 and 1970,^82-84 reemerging in the late 1990's.

West Nile virus strikingly exemplifies how unpredictably flaviviruses can emerge in naïve populations. In the United States, WNV lineage 1 was introduced in New York City in 1999, producing large and dramatic outbreaks in humans and horses. It rapidly spread throughout the country, causing more than 30 000 cases and 1200 deaths in humans and more than 24 000 cases in horses within a 10-year period.⁸² In contrast, Europe epidemics were irregular and limited in time and space from 1996 to 2010.

Until 2004, only lineages 1 and 3 WNV strains had been found in Europe. A first WNV lineage 2 strain was initially detected in Hungary in 2004,^{85, 86} with a noticeable increase in WNV transmission and outbreaks in Europe since the 2010 s associated with the spread of these WNV lineage 2 strains (Table 1).^{1, 88} Another WNV lineage 2 strain, first detected in 2004 in Southern Russia,⁹⁸ has also been occasionally reported in Europe.

In Europe the most important transmission wave occurred in 2018. At the end of 2018, a total of 1503 human confirmed cases were reported in 11 countries of the EU, of which some experienced the first autochthonous infections.⁹⁵ Reports from the ECDC also indicated a high transmission among horses with 285 outbreaks⁹⁹ (Table 2). Independently of the spread of the dominant lineage 2 strains, lineage 1 strains can be still responsible for local outbreaks like the 1 in Spain in 2020 (77 human and 137 equine cases).^{101, 102}

Usutu virus is thought to have been introduced into Europe multiple times through bird migration, starting in the 1950s.^{71, 103} In Europe, the first cases of USUV-associated large-scale deaths of wild birds were described in Italy in 1996 and in Austria in 2001.⁸⁹ Within 2 decades, USUV has spread all over Europe, except for the Baltic countries and Scandinavia, and in 2020, USUV emerged in the United Kingdom.¹⁰⁴ Viruses from African lineages were recently detected in Germany¹⁰⁵ and the Camargue area of France.¹⁰⁶

Tick-borne encephalitis virus is the most important human tick-borne pathogen in Europe and Asia. Temporal and geographical distribution of seropositivity in horse populations is highly variable in Europe^{9, 39, 107, 108} (Figure 4). Horses seem to be less sensitive to the pathogen; even with an unexpectedly high infection rate between 20% and 30% of TBEV in Austria, Germany and Lithuania,^{40, 109-111} equine clinical disease is rarely identified.^{42, 43, 111}

Based on epidemiological investigations, LIV distribution area was initially limited to the British Isles (particularly in Scotland, Cumbria, Wales, Devon and Ireland), but seroconversion or clinical cases in sheep due to LIV-like viruses have been reported during the last decade in Norway, Denmark, and Spain.^{9, 54, 56, 112} A limited serological survey in Ireland disclosed a positive rate of 10.6%, indicating a moderate infection rate in animals of mixed breed and of different environmental backgrounds.⁵⁶ Suspected equine neuroinvasive cases have been reported from clusters in the British Isles.¹¹³

Equine hepacivirus is globally distributed with reported cases in all continents except for Antarctica (Figure 5).^{3, 58, 114-118} Its prevalence was reported to be between 2% and 40% on the nucleic acid level while the seroprevalence ranged between 22% and 84% in different equine populations.^{12, 115, 116, 118-120} Noteworthy, EqHV, so far, does not cluster in different geographical genotypes, and all deposited sequences show high similarity, in contrast to the high diversity observed in HCV.¹²¹

4.1 DIAGNOSIS OF WEST NILE VIRUS INFECTION IN EQUIDS

4.1.1 Antemortem diagnosis of WNV infection

In Europe the following diseases should be included in differential diagnosis: other flaviviruses (USUV, TBEV, LIV) which have similar seasonal (although TBE usually diagnosed early summer) and clinical presentations and cross-react in most of the diagnostic tests. Apart from equine herpesvirus myeloencephalopathy, which is endemic in Europe, rabies and Borna-virus infection can occur in certain geographical regions. Verminous meningoencephalomyelitis, bacterial meningitis, botulism, toxicosis and trauma may lead to similar acute neurologic signs as WNND. Horses with cervical vertebral malformation or equine degenerative myeloencephalopathy usually show more age-related and chronic disease progression. Autochthonous cases caused by Alphaviruses or equine protozoal myeloencephalitis have not been diagnosed so far in Europe.

A suspected diagnosis of WNV infection is generally considered based on origin of the patient (horse residing in an endemic area for WNV, Figure 4), time of year, clinical presentation of the horse and vaccination history (ie, no or inadequate vaccination history against WNV).^{17, 20, 122} Ancillary diagnostic tests such as complete blood count (CBC), biochemical analysis and cerebrospinal fluid (CSF) analysis could help rule in or rule out other neurological or systemic causes. Similar to other viral infections, horses can display a mild, absolute lymphopenia. Biochemical abnormalities might suggest organ-related conditions causing neurological deficits such as liver disease. Muscle enzymes can be elevated in horses experiencing trauma or prolonged periods of recumbency. CSF analysis is often nonspecific and variable with elevated nucleated cell count (mononuclear or neutrophilic pleocytosis) and/or protein concentration and mild xanthochromia.^{123, 124} The antemortem detection of WNV in blood and/or CSF via culture and qPCR is rarely achieved because of the short-lived and low-level viremia reported in equids.^{34, 125}

Neutralizing antibodies have a critical role in the long-term protection from disease and, presently, their measurement provides the best correlate of flavivirus immunity and can be used for WNV field surveillance and for clinical diagnostics.¹²⁶ The flavivirus envelope (E) protein is the major target of virus neutralizing antibodies. The flaviviruses are antigenically related and broad serological cross-reactions in general and cross-neutralizations in particular are observed. Their extent and duration are strongly dependent on amino acid similarity in the E-protein which ranges from 40% to 44% for unrelated flaviviruses and 60% to 70% within closely related flaviviruses.¹²⁷

Even though there is a problem of cross-reactions with most of the serological tests, serology still provides the mainstay of presumptive WNV antemortem diagnosis (Figure 6). West Nile virus IgM-specific antibodies are often targeted to document recent infection since these antibodies rise sharply during the first few days of clinical disease and can be detected for about 4 to 6 weeks. However, because IgM antibodies can be detected up to 52 days after vaccination with either recombinant or inactivated vaccines, the serological results always need to be interpreted in the context of recent immunization.^{128, 129} From a diagnostic standpoint, the detection of intrathecally-derived IgM antibodies to WNV in CSF is even more conclusive to support a WNV infection, if there is no evidence of blood contamination or loss of integrity of the blood-brain barrier.¹³⁰ The serological test of choice offered by many veterinary diagnostic laboratories is the IgM WNV capture ELISA (MAC). The detection of anti-WNV IgM via MAC-ELISA is more specific and sensitive than the IgG assays for currently circulating European viruses, so the diagnosis of WNV infection often only requires a single test on serum, CSF or both.^{131, 132} However, cross-reactivity with other viruses of the JEV group like JEV is possible and such the reliability in areas with cocirculating cross-reacting viruses is questionable.^{133, 134} In Europe, circulating closely related flaviviruses like TBEV, USUV, and possibly LIV, induce antibodies that generate positive results in rapid serological diagnostic tests detecting IgG.¹³⁵ The gold standard confirmatory serological test in a nonvaccinated horse is thus based on the detection of serum neutralizing antibodies. All positive serological results using rapid assays should be confirmed by the more specific virus neutralization test (VNT) with the viruses known to circulate in the area (Figure 6). In nonvaccinated horses, a 4-fold change in paired neutralizing antibody titers is confirmatory of a diagnosis of WNND. Serum neutralizing antibodies can be measured by 1 of 2 formats, the plaque reduction neutralization test (PRNT) or the microwell (SN) neutralization test. The PRNT is the most commonly used neutralizing antibody test because the results are less influenced by individual differences between samples.¹²⁶ Since vaccination induces formation of neutralizing antibody to the E protein of the virus, the interpretation of serological results using PRNT can be more challenging. Additional tests, such as the microsphere immunoassay, hemagglutination inhibition test and indirect fluorescent antibody test, have been developed and are often used for the surveillance of WNV in various animal species.^136-138

4.2 Diagnosis of TBEV and USUV

Diagnosis of infection and disease caused by TBEV and USUV follows the same algorithm as previously described in the WNV section.

4.3 Diagnosis of LIV

In the field, serology plays a key role in confirming infection. The hemagglutination inhibition test can be used as a simple means of monitoring seroconversion in man and livestock.^{143, 144} Because LIV is cytolytic in tissue culture, a plaque reduction neutralization test using LIV can also be used to measure antiviral titers.⁵⁴ There is no data available in horses whether neutralizing antibodies against LIV can cross-react with other flaviviruses but it must be assumed based on some limited data in humans.¹⁴⁵

4.4 Diagnosis of EqHV

EqHV RNA can be isolated from serum or liver specimens and virus copy number can subsequently be quantified via quantitative RT-PCR. For reliable virus detection, highly conserved regions such as the 5′ untranslated region (UTR) are targeted.¹⁴⁶ Moreover, the Luciferase Immunoprecipitation System (LIPS) allows semiquantitative serologic detection of anti-EqHV specific IgG antibodies, although this is available on a research basis only at this time. For the detection of previous hepacivirus infection, the NS3 protease is a suitable target, since it is highly conserved.¹⁴⁶ Horses are seropositive during acute hepatitis and persistent infection.^{13, 14}

5.1 Flaviviruses—WNV, USUV, TBEV, LIV

In horses, survival rate for WNV encephalitis is high compared with other infectious encephalitides (55%-70%).^{21, 22, 33, 35} Treatment of WNND (and neuroinvasive diseases caused by TBEV) is mainly supportive. Nonsteroidal anti-inflammatory agents (flunixin-meglumine: 1.1 mg / kg, q12h IV) are often used for 3 to 7 days after onset of clinical signs to reduce inflammation and relieve pain. The use of corticosteroids is controversial because of their immunosuppressive effect that might increase viremia.¹⁴⁷ On the other hand, it can be hypothesized that the immunomodulatory effects of corticosteroids might hold benefits by reducing the amount of immune-mediated inflammation of the CNS. There is no scientific evidence supporting the use of corticosteroids, but also there are no contraindications and short-acting glucocorticoids (dexamethasone sodium: 0.05-0.1 mg/kg q24h IV) are often administered in cases of quickly deteriorating or recumbent animals. Antiviral agents and immunoglobulin therapy have been tested in studies mainly in humans and other species but did not gain widespread use due to limited data on their efficacy.¹⁴⁸

There is currently no treatment available against EqHV infections in equids. There are many direct-acting antiviral treatments for the closely related HCV, at least 1 of which, sofosbuvir, has been predicted to also bind EqHV by computer modeling.¹⁴⁹ Although it is possible that these treatments could be adapted for equine use, important hurdles remain, among these ethical concerns in sourcing such medications for equine use when they are not easily obtained for human patients.

6.1 Vaccination schedule and recommendations

West Nile virus vaccines are usually recommended from 5 to 6 months of age, depending on the WNV vaccines used, with annual boost immunization (usually recommended in spring before the mosquito season). The frequency of boost immunization could be adjusted based on the epidemiological situation (eg, semiannual or more frequently in case of active circulation, at the start of the mosquito season). Vaccination of pregnant mares is recommended before the breeding season, with a boost-immunization a few weeks before foaling to provide passive immunity to foals through colostrum.

The impact of preexisting immunity (either maternal derived immunity or previous immunization) on subsequent immunization has not been fully determined.¹⁵⁵ Horses that have been subclinically infected or recovered from clinical disease might have long-term immunity, but there is only limited, nonpublished data about the topic and thus it is recommended to vaccinate these horses.

Recommendation for WNV vaccination will vary from country to country. For example, the HBLB Code of conduct recommend WNV vaccination for horses traveling to countries with active WNV circulation¹⁵⁷ while the AAEP recommends immunization for all horses in North America.¹⁵⁸ In general, vaccination is recommended in endemic areas.

There are no vaccines licensed for horses against TBEV, USUV, LIV or EqHV. To date, there is no available scientific data that reports potential cross-protection induced by a flavivirus vaccine against infection with heterologous flaviviruses in horses.

7.1 Syndromic surveillance of West Nile virus activity

Horses as nonhuman vertebrate hosts are particularly sensitive to WNV, and among clinically affected horses, approximately 10% present with neurological disorders as compared to 1% of humans^{159, 160} rendering its detection in equids highly pertinent in a public health perspective.¹⁶¹ Considering that horses are spatially dispersed within human populations, and equine WNV infections typically precede human infections,^{160, 162, 163} a syndromic surveillance system based on the analysis of these data could be especially useful in serving as early warning for possible human viral infections.¹⁶⁴ However, temporal distribution of previous outbreaks does not always support the hypothesis that clusters of equine cases precede human cases.^{27, 33, 165, 166} This situation raises concerns about the suitability of passive WNV surveillance strategies in horses.

It is suspected that, by now, large numbers of horses have been exposed to WNV in many parts of Europe and have not developed clinical signs, while developing protective immunity. This, in combination with widespread vaccination, should eventually decrease the pool of susceptible horses and decrease their role in syndromic surveillance systems. On the other hand, clinical findings in horses might be of help for syndromic surveillance for WNV activity in countries so far considered to be free of WNV.¹⁶⁷

7.2 EqHV as a model virus

EqHV is the closest known relative to HCV, an important human pathogen. Since HCV and EqHV share common features, EqHV is also an interesting surrogate model to study hepacivirus immune responses and pathogenesis.