American alligators are capable of West Nile virus amplification, mosquito infection and transmission.

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

Mosquito-Independent Transmission of West Nile virus in Farmed Saltwater Crocodiles (Crocodylus porosus).

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.

Crocodilepox Virus Evolutionary Genomics Supports Observed Poxvirus Infection Dynamics on Saltwater Crocodile (Crocodylus porosus).

Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2's SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting.

Adenoviral hepatitis in two Nile crocodile (Crocodylus niloticus) hatchlings from South Africa.

Adenoviral infections may cause mild to severe morbidity or fatality in a large array of animal species. In crocodilians, hatchlings under 5 months of age are usually affected. However, there is a paucity of information on actual incidences in hatchlings originating from South Africa. Two cases of adenoviral hepatitis in crocodile hatchlings about 2 weeks old, bred on a commercial farm in South Africa, are described. Both hatchlings showed typical clinical signs of hepatitis. The identification of intranuclear inclusion bodies in the liver was used to differentiate between adenoviral hepatitis and chlamydial hepatitis. Although vertical transmission has never been proven in crocodiles, the young age of the affected hatchlings raises the possibility of vertical transmission. The lack of epidemiological information on adenoviral hepatitis in crocodiles highlights the need for further characterisation of the virus and targeted surveillance.

First evidence of Kunjin strain of West Nile virus associated with saltwater crocodile (Crocodylus porosus) skin lesions.

Recently, the Kunjin strain of West Nile virus (WNVKUN ) has been detected using qRT-PCR in belly skin lesions of farmed juvenile saltwater crocodiles. This follows an established association between similar lesions and West Nile virus in American alligators. The lesions present as cutaneous lymphohistiocytic aggregates in the dermal layers of both species. While these lesion do not create an obvious defect on the live crocodile, upon tanning the lesion area collapses and does not uptake the dye evenly, thus reducing its aesthetic appeal. As a result, skins are being rejected jeopardising the economic viability of the Australian crocodile industry. Over 50 skin lesions have since been confirmed as WNVKUN -positive and preliminary evidence of lesion restructuring is presented. Horizontal transmission of WNVKUN by mosquitoes is well-established but other transmission routes, such as ingestion and cloacal shedding, need further evaluation. An infection trial is currently underway to ensure WNVKUN is the causative agent of these skin lesions.

Dermatological conditions of farmed Crocodilians: A review of pathogenic agents and their proposed impact on skin quality.

The control of pathogens that target crocodilian skin is essential to the long-term success and sustainability of intensive farming operations worldwide. To understand the impact these pathogens may have on the skin, a brief overview of skin histology is given. A review of the known viral, bacterial, fungal and helminth taxa associated with skin conditions in commercially significant crocodilian species is presented. Best management practices are discussed, with an emphasis on addressing extrinsic factors that influence transmission and pathogenicity. It is argued that, in the past, reduced immune function arising from inadequate thermal regulation was the leading cause of skin disease in captive crocodilians. Consequently, innovations such as temperature control, coupled with the adoption of more stringent hygiene standards, have greatly reduced the prevalence of many infectious skin conditions in intensively farmed populations. However, despite improvements in animal husbandry and disease management, viral pathogens such as West Nile virus, herpesvirus and poxvirus continue to afflict crocodilians in modern captive production systems.

Molecular characterization of the first saltwater crocodilepox virus genome sequences from the world's largest living member of the Crocodylia.

Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.

Neutralizing antibodies for orthobunyaviruses in Pantanal, Brazil.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

Impact of poxvirus lesions on saltwater crocodile (Crocodylus porosus) skins.

Cutaneous poxvirus infections are common in several crocodilian species and are of importance in crocodile farming due to their potential impact on the tanned hide. To confirm poxvirus infection and understand the impact on saltwater crocodile (Crocodylus porosus) skin, fourteen animals from different age groups (five hatchlings, five yearlings and four grow-outs) were selected based on a criterion of ten poxvirus-like lesions per animal. One lesion on each animal was extruded for genetic analysis and transmission electron microscopy. Both methods confirmed poxvirus so the remainder of lesions were re-examined every six weeks over a 24 week study period. Each lesion went through four distinct phases: early active, active, expulsion and healing. To understand how these lesions impact on the final skin product, one crocodile from each age group was euthanised and the lesions examined. Using standard skin grading techniques (light-table), the early phase (early active - expulsion) lesions were all translucent and would lead to downgrading of the skin or, at worst, rendering them unsaleable. At the later stages of healing, the translucency reduces. Histological examination of the phases confirm that the basement membrane is not breached by the infection further indicating that poxvirus lesions, given enough time, will eventually have no detrimental effect on skin quality. This is obviously dependent upon no more lesions developing in the interim.

Molecular identification of three novel herpesviruses found in Australian farmed saltwater crocodiles (Crocodylus porosus) and Australian captive freshwater crocodiles (Crocodylus johnstoni).

As part of a larger investigation into three emerging disease syndromes highlighted by conjunctivitis and pharyngitis, systemic lymphoid proliferation and encephalitis, and lymphonodular skin infiltrates in farmed saltwater crocodiles (Crocodylus porosus) and one emerging syndrome of systemic lymphoid proliferation in captive freshwater crocodiles (Crocodylus johnstoni), cytopathic effects (CPE), including syncytial cell formation, were observed in primary crocodile cell lines exposed to clarified tissue homogenates from affected crocodiles. Ten cell cultures with CPE were then screened for herpesviruses using two broadly-reactive herpesvirus PCRs. Amplicons were obtained from 9 of 10 cell cultures and were sequenced. Three novel herpesviruses were discovered and the phylogenetic analysis of these viruses showed there was a 63% Bayesian posterior probability value supporting these viruses clustering with the subfamily Alphaherpesvirinae, and 100% posterior probability of clustering with a clade containing the Alphaherpesvirinae and other unassigned reptile herpesviruses. It is proposed that they are named Crocodyline herpesvirus (CrHV) 1, 2 and 3. CrHV1 and 2 were only isolated from saltwater crocodiles and CrHV3 was only isolated from freshwater crocodiles. A duplex PCR was designed that was able to detect these herpesviruses in formalin-fixed paraffin-embedded tissues, a sample type that neither of the broadly-reactive PCRs was able to detect these herpesviruses in. This work describes the isolation, molecular detection and phylogeny of these novel herpesviruses but the association that they have with the emerging disease syndromes requires further investigation.

Surveys of endogenous retroviruses (ERVs) in the freshwater crocodile (Crocodylus johnstoni) suggest that ERVs in Crocodylus spp. vary between species.

Endogenous retroviruses (ERVs) are one of many families of transposable elements present in vertebrate genomes. We have examined the ERV complement of the freshwater crocodile (Crocodylus johnstoni) in order to investigate the diversity of ERVs present and possibility of ERV or retroviral activity in a diseased individual of this species. Amplification and sequencing of the highly conserved retroviral pro-pol domains revealed high levels of sequence variation in these ERVs. Phylogenetic analyses of these ERVs and those previously identified in other crocodilian species suggest that although many crocodilians share the same ERV lineages, the relative numbers of retroelement insertions from each of these lineages may vary greatly between species. The data generated in this study provide evidence for the presence of a unique and varied complement of ERVs in crocodilians. This study has also demonstrated the presence of species-specific evolution in ancient retroviral infections.

Comparative genome analyses reveal distinct structure in the saltwater crocodile MHC.

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2-6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

Early mesozoic coexistence of amniotes and hepadnaviridae.

Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.

Evolution and gene capture in ancient endogenous retroviruses - insights from the crocodilian genomes.

BACKGROUND: Crocodilians are thought to be hosts to a diverse and divergent complement of endogenous retroviruses (ERVs) but a comprehensive investigation is yet to be performed. The recent sequencing of three crocodilian genomes provides an opportunity for a more detailed and accurate representation of the ERV diversity that is present in these species. Here we investigate the diversity, distribution and evolution of ERVs from the genomes of three key crocodilian species, and outline the key processes driving crocodilian ERV proliferation and evolution. RESULTS: ERVs and ERV related sequences make up less than 2% of crocodilian genomes. We recovered and described 45 ERV groups within the three crocodilian genomes, many of which are species specific. We have also revealed a new class of ERV, ERV4, which appears to be common to crocodilians and turtles, and currently has no characterised exogenous counterpart. For the first time, we formally describe the characteristics of this ERV class and its classification relative to other recognised ERV and retroviral classes. This class shares some sequence similarity and sequence characteristics with ERV3, although it is phylogenetically distinct from the other ERV classes. We have also identified two instances of gene capture by crocodilian ERVs, one of which, the capture of a host KIT-ligand mRNA has occurred without the loss of an ERV domain. CONCLUSIONS: This study indicates that crocodilian ERVs comprise a wide variety of lineages, many of which appear to reflect ancient infections. In particular, ERV4 appears to have a limited host range, with current data suggesting that it is confined to crocodilians and some lineages of turtles. Also of interest are two ERV groups that demonstrate evidence of host gene capture. This study provides a framework to facilitate further studies into non-mammalian vertebrates and highlights the need for further studies into such species.

Serological evidence of widespread circulation of West Nile virus and other flaviviruses in equines of the Pantanal, Brazil.

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT90) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.

Antibodies to West Nile virus in wild and farmed crocodiles in southeastern Mexico.

Surveillance for evidence of West Nile virus (WNV) infection in Morelet's crocodiles (Crocodylus moreletii) was conducted in Campeche State, Mexico, in 2007. Sera from 62 crocodiles (32 free-ranging and 30 captive) were assayed for antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay. Antibodies to WNV were detected in 13 (41%) wild and nine (30%) captive crocodiles, and the overall antibody prevalence was 35%. Although evidence of WNV infection in captive crocodiles has been reported in Mexico, we provide the first evidence of WNV exposure in wild crocodiles in Mexico.

Neutralising antibodies for West Nile virus in horses from Brazilian Pantanal.

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of ≥ four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.

Identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed Nile crocodiles, Crocodylus niloticus.

When large numbers of crocodile skins were downgraded because of the presence of small pin prick-like holes, collapsed epidermal cysts were found deep in the dermis of juvenile crocodiles while forming cysts were observed in hatchlings. Histopathology of these forming cysts showed the presence of intracytoplasmic inclusions in proliferating and ballooning epidermal cells. Pox virions were seen in electron microscope preparations made from the scabs of such early lesions. The partial sequencing of virus material from scrapings of these lesions and comparison of it with the published sequence of crocodile poxvirus showed the virus associated with the deep lesions to be closely related, but different. To differentiate between the two forms of crocodile pox infection it is suggested that the previously known form should be called "classical crocodile pox" and the newly discovered form "atypical crocodile pox". The application of strict hygiene measures brought about a decline in the percentage of downgraded skins.

Association of West Nile virus with lymphohistiocytic proliferative cutaneous lesions in American alligators (Alligator mississippiensis) detected by RT-PCR.

West Nile virus (WNV) is known to affect captive populations of alligators and, in some instances, cause significant mortalities. Alligators have been shown to amplify the virus, serve as a reservoir host, and even represent a source of infection for humans. This study describes a cutaneous manifestation of WNV in captive-reared American alligators (Alligator mississippiensis), previously described as lymphohistiocytic proliferative syndrome of alligators (LPSA), based on the findings of gross examination, histopathologic evaluation, WNV antibody testing, and WNV reverse transcriptase polymerase chain reaction (RT-PCR). Forty alligators with LPSA and 41 controls were examined. There was a significant difference (P = 0.01(-21)) in the WNV serostatus between the treatment group (100%) and the control group (0%, 95% CI: 0-7.3%). In the treatment group, 97.5% (39/40) (95% CI: 92.7-102.3%) of the LPSA skin lesions were positive for WNV via RT-PCR. Of the skin sections within the treatment group that had no LPSA lesions, 7.5% (3/40) (95% CI: 0-15.7%) were positive for WNV. In the control group, all of the skin samples were negative for WNV (41/41) (0%; 95% CI: 0-7.3%). The LPSA skin lesions were significantly more likely to be WNV positive by RT-PCR when compared to control animals (P = 0.07(-20)) and normal skin sections from affected animals (P = 0.08(-16)). There was no significant difference in the WNV RT-PCR results between control animals and normal skin sections from affected animals (P = 0.24). These findings suggest that LPSA is a cutaneous manifestation of WNV in alligators.

Do crocodilians get the flu? Looking for influenza A in captive crocodilians.

It is well established that several wild aquatic bird species serve as reservoirs for the influenza A virus. It has also been shown that the influenza A virus can be transmitted to mammalian species such as tigers and domestic cats and dogs through ingestion of infected birds. Another group of animals that should also be considered as potential hosts for the influenza A virus are the crocodilians. Many crocodilian species share aquatic environments with wild birds that are known to harbor influenza viruses. In addition, many large crocodilians utilize birds as a significant food source. Given these factors in addition to the close taxonomic proximity of aves to the crocodilians, it is feasible to ask whether crocodilian species may also harbor the influenza A virus. Here we analyzed 37 captive crocodilians from two locations in Florida (plus 5 wild bird fecal-samples from their habitat) to detect the presence of influenza A virus. Several sample types were examined. Real-time RT-PCR tests targeting the influenza A matrix gene were positive for four individual crocodilians--Alligator sinensis, Paleosuchus trigonatus, Caiman latirostris and Crocodylus niloticus. Of the seven serum samples tested with the avian influenza virus agar gel immunodiffusion assay, three showed a nonspecific reaction to the avian influenza virus antigen-A. sinensis, P. trigonatus and C. niloticus (C. latirostris was not tested). Viable virus could not be recovered from RT-PCR-positive samples, although this is consistent with previous attempts at viral isolation in embryonated chicken eggs with crocodilian viruses.