Detection of SARS-CoV-2 RNA in a Zoo-Kept Red Fox (Vulpes vulpes).

Many different animal species are susceptible to SARS-CoV-2, including a few Canidae (domestic dog and raccoon dog). So far, only experimental evidence is available concerning SARS-CoV-2 infections in red foxes (Vulpes vulpes). This is the first report of SARS-CoV-2 RNA detection in a sample from a red fox. The RT-qPCR-positive fox was zoo-kept together with another fox and two bears in the Swiss Canton of Zurich. Combined material from a conjunctival and nasal swab collected for canine distemper virus diagnostics tested positive for SARS-CoV-2 RNA with Ct values of 36.9 (E gene assay) and 35.7 (RdRp gene assay). The sample was analysed for SARS-CoV-2 within a research project testing residual routine diagnostic samples from different animal species submitted between spring 2020 and December 2022 to improve knowledge on SARS-CoV-2 infections within different animal species and investigate their potential role in a One Health context. Within this project, 246 samples from 153 different animals from Swiss zoos and other wild animal species all tested SARS-CoV-2 RT-qPCR and/or serologically negative so far, except for the reported fox. The source of SARS-CoV-2 in the fox is unknown. The fox disappeared within the naturally structured enclosure, and the cadaver was not found. No further control measures were undertaken.

Detection of SARS-CoV-2 Delta Variant (B.1.617.2) in Domestic Dogs and Zoo Tigers in England and Jersey during 2021.

Reverse zoonotic transmission events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described since the start of the pandemic, and the World Organisation for Animal Health (WOAH) designated the detection of SARS-CoV-2 in animals a reportable disease. Eighteen domestic and zoo animals in Great Britain and Jersey were tested by APHA for SARS-CoV-2 during 2020-2023. One domestic cat (Felis catus), three domestic dogs (Canis lupus familiaris), and three Amur tigers (Panthera tigris altaica) from a zoo were confirmed positive during 2020-2021 and reported to the WOAH. All seven positive animals were linked with known SARS-CoV-2 positive human contacts. Characterisation of the SARS-CoV-2 variants by genome sequencing indicated that the cat was infected with an early SARS-CoV-2 lineage. The three dogs and three tigers were infected with the SARS-CoV-2 Delta variant of concern (B.1.617.2). The role of non-human species in the onward transmission and emergence of new variants of SARS-CoV-2 remain poorly defined. Continued surveillance of SARS-CoV-2 in relevant domestic and captive animal species with high levels of human contact is important to monitor transmission at the human-animal interface and to assess their role as potential animal reservoirs.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Viruses in sanctuary chimpanzees across Africa.

Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild-born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long-term conservation strategies. We used metagenomic next-generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other "atypical" or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas-Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector-borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood-borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.

Effects of between and within Herd Moves on Elephant Endotheliotropic Herpesvirus (EEHV) Recrudescence and Shedding in Captive Asian Elephants (Elephas maximus).

Haemorrhagic disease associated with elephant endotheliotropic herpesvirus (Elephantid herpesvirus, EEHV) infections is the leading cause of death for Asian elephant (Elephas maximus) calves. This study assessed the effect of captive herd management on EEHV shedding, as evidence of latent infection reactivation, focusing on: (1) the influence of social change on the odds of recrudescence; (2) the respective effects of between and within herd moves; and (3) characteristics of recrudescent viral shedding. Trunk and conjunctival swabs (n = 165) were obtained from six elephants at an EAZA-accredited zoo, collected during a period of social stability, and at times of social change. Longitudinal sampling took place at times of moving two bulls out of the collection and one new bull into an adjacent enclosure to the cow herd (between herd moves), and during a period of mixing this new bull with the cow herd to facilitate mating (within herd moves). Quantitative PCR was employed to detect EEHV 1a/b, 4a/b, and EF-1-α (housekeeping gene). Generalised estimating equations determined EEHV recrudescence odds ratios (OR) and relative viral DNA load. Sixteen EEHV 1a/b shedding events occurred, but no EEHV 4a/b was detected. All management-derived social changes promoted recrudescence (social change OR = 3.27, 95% CI = 0.412-26, p = 0.262; and between herd moves OR = 1.6, 95% CI = 0.178-14.4, p = 0.675), though within herd movements posed the most significant increase of EEHV reactivation odds (OR = 6.86, 95% CI = 0.823-57.1, p = 0.075) and demonstrated the strongest relative influence (post hoc Tukey test p = 0.0425). Shedding onset and magnitude ranged from six to 54 days and from 3.59 to 11.09 ΔCts. Differing challenges are associated with between and within herd movements, which can promote recrudescence and should be considered an exposure risk to naïve elephants.

Primary Infection May Be an Underlying Factor Contributing to Lethal Hemorrhagic Disease Caused by Elephant Endotheliotropic Herpesvirus 3 in African Elephants (Loxodonta africana).

Distinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection. IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations.

Understanding the prevalence of SARS-CoV-2 (COVID-19) exposure in companion, captive, wild, and farmed animals.

Several animal species, including ferrets, hamsters, monkeys, and raccoon dogs, have been shown to be susceptible to experimental infection by the human severe acute respiratory syndrome coronaviruses, such as SARS-CoV and SARS-CoV-2, which were responsible for the 2003 SARS outbreak and the 2019 coronavirus disease (COVID-19) pandemic, respectively. Emerging studies have shown that SARS-CoV-2 natural infection of pet dogs and cats is also possible, but its prevalence is not fully understood. Experimentally, it has been demonstrated that SARS-CoV-2 replicates more efficiently in cats than in dogs and that cats can transmit the virus through aerosols. With approximately 470 million pet dogs and 370 million pet cats cohabitating with their human owners worldwide, the finding of natural SARS-CoV-2 infection in these household pets has important implications for potential zoonotic transmission events during the COVID-19 pandemic as well as future SARS-related outbreaks. Here, we describe some of the ongoing worldwide surveillance efforts to assess the prevalence of SARS-CoV-2 exposure in companion, captive, wild, and farmed animals, as well as provide some perspectives on these efforts including the intra- and inter-species coronavirus transmissions, evolution, and their implications on the human-animal interface along with public health. Some ongoing efforts to develop and implement a new COVID-19 vaccine for animals are also discussed. Surveillance initiatives to track SARS-CoV-2 exposures in animals are necessary to accurately determine their impact on veterinary and human health, as well as define potential reservoir sources of the virus and its evolutionary and transmission dynamics.

A Sentinel Serological Study in Selected Zoo Animals to Assess Early Detection of West Nile and Usutu Virus Circulation in Slovenia.

Monitoring infectious diseases is a crucial part of preventive veterinary medicine in zoological collections. This zoo environment contains a great variety of animal species that are in contact with wildlife species as a potential source of infectious diseases. Wild birds may be a source of West Nile virus (WNV) and Usutu (USUV) virus, which are both emerging pathogens of rising concern. The aim of this study was to use zoo animals as sentinels for the early detection of WNV and USUV in Slovenia. In total, 501 sera from 261 animals of 84 animal species (including birds, rodents, lagomorphs, carnivores, ungulates, reptiles, equids, and primates) collected for 17 years (2002-2018) were tested for antibodies to WNV and USUV. Antibodies to WNV were detected by indirect immunofluorescence tests in 16 (6.1%) of 261 animals representing 10 species, which were sampled prior to the first active cases of WNV described in 2018 in Slovenia in humans, a horse, and a hooded crow (Corvus cornix). Antibodies to USUV were detected in 14 out of 261 animals tested (5.4%) that were positive prior to the first positive cases of USUV infection in common blackbirds (Turdus merula) in Slovenia. The study illustrates the value of zoological collections as a predictor of future emerging diseases.

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China.

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country.

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Herpesvirus surveillance and discovery in zoo-housed ruminants.

Gammaherpesvirus infections are ubiquitous in captive and free-ranging ruminants and are associated with a variety of clinical diseases ranging from subclinical or mild inflammatory syndromes to fatal diseases such as malignant catarrhal fever. Gammaherpesvirus infections have been fully characterized in only a few ruminant species, and the overall diversity, host range, and biologic effects of most are not known. This study investigated the presence and host distribution of gammaherpesviruses in ruminant species at two facilities, the San Diego Zoo and San Diego Zoo Safari Park. We tested antemortem (blood, nasal or oropharyngeal swabs) or postmortem (internal organs) samples from 715 healthy or diseased ruminants representing 96 species and subspecies, using a consensus-based herpesvirus PCR for a segment of the DNA polymerase (DPOL) gene. Among the 715 animals tested, 161 (22.5%) were PCR and sequencing positive for herpesvirus, while only 11 (6.83%) of the PCR positive animals showed clinical signs of malignant catarrhal fever. Forty-four DPOL genotypes were identified of which only 10 have been reported in GenBank. The data describe viral diversity within species and individuals, identify host ranges of potential new viruses, and address the proclivity and consequences of interspecies transmission during management practices in zoological parks. The discovery of new viruses with wide host ranges and presence of co-infection within individual animals also suggest that the evolutionary processes influencing Gammaherpesvirus diversity are more complex than previously recognized.

Feline Panleukopenia Virus With G299E Substitution in the VP2 Protein First Identified From a Captive Giant Panda in China.

A feline panleukopenia virus (FPV), Giant panda/CD/2018, was isolated from a captive giant panda with mild diarrhea in 2018 in Chengdu, China, and further identified via indirect immunofluorescence assay (IFA), transmission electron microscopy (TEM) observation, and genetic analysis. Phylogenetic analysis based on the complete VP2 nucleotide sequences showed that it shared high homology with Chinese FPV isolates and grouped within FPV cluster 1. One unique substitution Gly(G)299Glu(E) in the capsid protein VP2 was first identified with Giant panda/CD/2018. The presence of the G299E substitution is notable as it is located on the top region of the interconnecting surface loop 3, which may be involved in controlling the host range and antigenicity of FPV. These findings first demonstrate that FPV with natural point mutation G299E in the VP2 gene is prevalent in giant panda and suggest that etiological surveillance and vaccination among all giant pandas are urgently needed to protect this endangered species against FPV infection.

Serpentovirus (Nidovirus) and Orthoreovirus Coinfection in Captive Veiled Chameleons (Chamaeleo calyptratus) with Respiratory Disease.

Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (Chamaeleo calyptratus). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40-50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80-90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Relatives of rubella virus in diverse mammals.

Since 1814, when rubella was first described, the origins of the disease and its causative agent, rubella virus (Matonaviridae: Rubivirus), have remained unclear1. Here we describe ruhugu virus and rustrela virus in Africa and Europe, respectively, which are, to our knowledge, the first known relatives of rubella virus. Ruhugu virus, which is the closest relative of rubella virus, was found in apparently healthy cyclops leaf-nosed bats (Hipposideros cyclops) in Uganda. Rustrela virus, which is an outgroup to the clade that comprises rubella and ruhugu viruses, was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice (Apodemus flavicollis) at and near the zoo. Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus2,3. The amino acid sequences of four putative B cell epitopes in the fusion (E1) protein of the rubella, ruhugu and rustrela viruses and two putative T cell epitopes in the capsid protein of the rubella and ruhugu viruses are moderately to highly conserved4-6. Modelling of E1 homotrimers in the post-fusion state predicts that ruhugu and rubella viruses have a similar capacity for fusion with the host-cell membrane5. Together, these findings show that some members of the family Matonaviridae can cross substantial barriers between host species and that rubella virus probably has a zoonotic origin. Our findings raise concerns about future zoonotic transmission of rubella-like viruses, but will facilitate comparative studies and animal models of rubella and congenital rubella syndrome.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.

Evidence of exposure to zoonotic flaviviruses in zoo mammals in Spain and their potential role as sentinel species.

A serosurvey was carried out to assess emerging flavivirus exposure in zoo mammals in Spain and to determine the dynamics of seropositivity in species that were longitudinally sampled during the study period. Sera from 570 zoo animals belonging to 120 mammal species were collected at ten zoos (A-J) in Spain between 2002 and 2019. Twenty-one of these animals, belonging to ten different species, were sampled longitudinally at four of the zoos during the study period. Antigenically-related flavivirus antibodies were detected in 19 (3.3 %; 95 %CI: 2.0-5.2) of the 570 animals analyzed using bELISA. Seropositivity was observed in ten (8.3 %) of the 120 species tested. Five (23.8 %) of the 21 animals sampled more than once presented seropositivity in all samplings whereas seroconversion was only observed in one white rhinoceros (Ceratotherium simum). Flavivirus antibodies were found at six of the ten sampled zoos and in consecutive years between 2008 and 2018. Virus neutralization tests confirmed West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) infection in ten (1.8 %; 95 %CI: 0.7-2.8), five (0.9 %; 95 %CI: 0.1-1.6) and one (0.2 %; 95 %CI: 0.0-0.5) animal, respectively. Antibodies against Meaban virus (0 %; 95 %CI: 0.0-0.7 %) were not found in the tested sera. The results demonstrate WNV, USUV and TBEV exposure in zoo mammals, which may be of public health and conservation concern. Seropositivity to WNV and USUV was detected in regions where these viruses have not been reported previously. Anti-WNV antibodies found in zoo animals sampled in 2009 point to WNV circulation at least one year before the first outbreaks were reported in horses and humans in Spain. Our results indicate that zoo mammals could be useful sentinel species for monitoring emerging flavivirus activity in urban areas.