Only one virus, Avipox, has been documented previously in wild birds in Hawaii. Using immunohistochemistry and PCR, we found that two native threatened Hawaiian Geese (Branta sandvicensis), one with multicentric histiocytoma and the other with toxoplasmosis, and one Laysan Albatross (Phoebastria immutabilis) with avian pox were infected with reticuloendotheliosis virus (REV). The virus was isolated from one of the geese by cell culture. Surveys of other Hawaiian geese with various pathologies, avian pox cases, and pox viral isolates using PCR failed to reveal REV, suggesting that the virus is uncommon, at least in samples examined. The full genome of the Gag, Pol, and Env genes were sequenced for all three infected birds and revealed geographic divergence of the Pol gene, suggesting it to be under strong selective pressure. Our finding of REV in Hawaii makes this only the second virus documented in native Hawaiian birds associated with pathology. Moreover, the presence of REV in a pelagic seabird is unusual. Future surveys should seek the reservoir of the virus in efforts to trace its origins.

Reticuloendotheliosis viruses (REVs) are tumor-inducing retroviruses within the avian leucosis group. In chickens (Gallus gallus), REV can cause lymphoid and reticulum cell neoplasia in various organs or runting and feather malformation in poults (Nair et al. 2013). The REVs are unique among avian retroviruses in that they apparently originated from a mammalian virus that infected the avian parasite Plasmodium lophurae and jumped from there to birds, hence their closer relatedness to mammalian retroviruses (Niewiadomska and Gifford 2013). The genome of REV also integrates into the genomes of Gallid herpesvirus 2, the cause of Marek's disease, and with the genome of Avipox, the cause of fowl pox (Nair et al. 2013). The ability of REV to integrate in these two viruses resulted in REV contamination of early vaccines against Marek's disease and fowl pox used in poultry production; this led to occasional outbreaks of REV in poultry flocks (Liu et al. 2009).

Reticuloendotheliosis virus infects 11 orders of birds (Drew 2007), but in contrast to other viruses of the leucosis group where virus infection is often associated with tumors (Payne and Nair 2012), REV-related tumor formation has primarily been seen in Galliformes, not in other bird taxa. The lack of association between REV and tumor formation in other bird taxa could partly be due to limited surveys for this virus in wild birds (Drew 2007). In nondomestic birds, REV has had important effects in recovery efforts for the endangered Attwater's Prairie Chicken (Tympanuchus cupido attwateri) for which significant mortality in captive flocks has been seen with >50% birds demonstrating persistent viremia (Drew et al. 1998; Zavala et al. 2006). Whereas REV is commonly observed in captive prairie chickens (Drew 2007), it is seldom (ca. 1% prevalence) observed in wild prairie chickens (Wiedenfeld et al. 2002). In Texas, US, PCR surveys in free-ranging Wild Turkeys (Meleagris gallopavo) for REV (Stewart et al. 2019), and serologic surveys in wild birds (Ferro et al. 2017), show that the virus in uncommon, with prevalence measured by PCR and serology at 1% and 5%, respectively.

Reticuloendotheliosis virus has a global distribution in poultry, but it has been rarely documented in the Pacific islands, with only one report from infected poultry in Papua New Guinea (Van Kammen 1982). This is possibly, in part, because of the limited available animal diagnostic capacity in the region (Brioudes et al. 2014). Also, it is well established that animals colonizing island ecosystems tend to leave their parasites behind (Lafferty et al. 2010), so the microbiota infecting native animals is expected to be relatively more depauperate. For instance, in Hawaii, the only virus known to circulate in wild birds is Avipox. This virus causes severe lesions and mortality in native honeycreepers (van Riper et al. 2002). In Laysan Albatross (Phoebastria immutabilis), infected chicks can recover from pox lesions, but they have decreased long-term survival as adults (VanderWerf and Young 2016). The probable true paucity of viruses in Hawaii is strengthened by a review of diagnostic records for 4,549 wild birds necropsied by the Honolulu Field Station since 1992, comprising 42 species of seabirds, 67 species of terrestrial birds, and 30 species of waterfowl for which 2,096 virus investigations (PCR, virus isolation) were done; the only virus ever detected was Avipox.

Prompted by the detection of REV in tissues of a native threatened Hawaiian (Nene) Goose (Branta sandvicensis) with tumors and a Laysan Albatross chick with avian pox, we screened archived tissues of geese and albatross for REV by PCR and immunohistochemistry (IHC). We also screened available pox virus isolates by PCR to see whether the virus integrated in Avipox.

Carcasses and pox virus isolates

Birds for necropsy were submitted to the US Geological Survey, National Wildlife Health Center, Honolulu Field Station (Honolulu, Hawaii, USA) as part of routine diagnostic procedures to determine causes of death in native Hawaiian birds (Work et al. 2015). Birds were weighed and received a complete external and internal examination, with representative tissues harvested in 10% formalin for histopathology and, as applicable, frozen. For histopathology, tissues were embedded in paraffin, sectioned at 5 µm, and stained with H&E. For REV testing, diagnoses were partitioned into two groups: 1) possible immunosuppressive condition including diagnoses such as neoplasia, inflammation, or infections with avian pox, bacteria, fungi, or toxoplasmosis, all of which could conceivably be enabled by underlying infections by immunosuppressive viruses such as REV (Drew 2007); or 2) nonimmunosuppressive diagnoses including trauma, emaciation, or undetermined (no gross or microscopic lesions explaining cause of death). Laboratory isolates of avian pox virus from various Hawaiian birds originated from cultures of tissue inocula into duck embryo fibroblasts as described previously (Atkinson et al. 2005).

Immunohistochemistry

Detection of REV by IHC followed the methods of Santos et al. (2008). Briefly, slides were deparaffinized in xylene, rehydrated through an ethanol series, and antigen heat retrieval was done using citrate buffer (pH 6). Slides were then equilibrated in buffer (Agilent, Santa Clara, California, USA), blocked with 5% casein for 30 min, and incubated overnight with chicken anti-REV (Charles Rivers Laboratories, Wilmington, Massachusetts, USA) diluted 1:5,000 in phosphate-buffered saline (PBS) at 4 C. Slides were then washed and incubated with biotinylated goat anti-chicken antiserum (Vector Laboratories, Burlingame, California, USA) diluted 1:5,000 in PBS for 1 h. Color development was done by incubating tissues with Vectastain ABC-AP (Vector Laboratories) for 30 min followed by washing and incubation with vector red substrate (Vector Laboratories). Negative controls consisted of reacting REV-positive tissues with secondary antibody only and reacting known negative tissues with both anti-REV and secondary antibody.

PCR assays

Endpoint PCR assays targeted the envelope (Env) region of REV by using primers as described previously (García et al. 2003). Briefly, DNA was extracted from tissues by using a DNEasy kit (Qiagen, Germantown, Maryland, USA) and PCRs were run with the following protocol to yield products ranging from 320 to 858 base-pair (bp) product: 94 C for 1 min followed by 35 cycles of 94 C for 30 s melting, 60 C for 30 s annealing, 72 C for 30 s elongation, with a final elongation of 72 C for 6 min. Amplicons were treated with Exosap-IT (Thermo Fisher, Waltham, Massachusetts, USA) and submitted to the University of Hawaii Advanced Studies in Genomic, Proteomics and Bioinformatics core facility, Honolulu, for Sanger sequencing.

We sequenced the complete Gag, Pol, and Env genes from three birds that were positive for REV by IHC and PCR. To do this, we aligned the complete sequences of REV comprising the following GenBank accessions: FJ496333.1, AY842951.1, DQ003591.1, DQ387450.1, FJ439119.1, FJ439120.1, GQ375848.1, GQ415646.2, JQ804915.1, JX912710.1, KF305089.1, KF709431.1, KJ756349.1, KJ909530.1, KJ909531.1, KU204702.1, KU204703.1, KU641115.1, KX278301.1, KY581581.1, MF185397.1, MF631845.1, MG471384.1, and NC_006934.1 and created a consensus sequence template with Bioedit (Hall 1999). Primers (Table 1) were designed against the template, and 27 approximately 600-bp overlapping regions were amplified in triplicate to get three forward and three reverse sequences for a total of six sequences for each region (Supplementary Material Table S1). For each region, the six sequences (three forward, three reverse) were aligned using MEGA7 (Tamura et al. 2013) and assembled to the Gag-Pol-Env cassette. Each gene was translated using EXPASY (Gasteiger et al. 2003), and proteins were blasted to the National Center for Biological Information protein database (Buchfink et al. 2015; National Center for Biotechnology Information 2021) to obtain candidate sequences for alignments and construction of phylogenetic trees.

Table 1

Virus variants and gene isoforms for reticuloendotheliosis virus sequenced from two free-living Hawaiian Geese (Branta sandvicensis) and one Laysan Albatross (Phoebastria immutabilis) from Hawaii, USA, partitioned by virus variant identification (ID) number and gene isoform ID at the nucleotide (Nuc) and amino acid (AA) levels, and GenBank ID.

Virus variants and gene isoforms for reticuloendotheliosis virus sequenced from two free-living Hawaiian Geese (Branta sandvicensis) and one Laysan Albatross (Phoebastria immutabilis) from Hawaii, USA, partitioned by virus variant identification (ID) number and gene isoform ID at the nucleotide (Nuc) and amino acid (AA) levels, and GenBank ID.
Virus variants and gene isoforms for reticuloendotheliosis virus sequenced from two free-living Hawaiian Geese (Branta sandvicensis) and one Laysan Albatross (Phoebastria immutabilis) from Hawaii, USA, partitioned by virus variant identification (ID) number and gene isoform ID at the nucleotide (Nuc) and amino acid (AA) levels, and GenBank ID.

For construction of phylogenetic trees, amino acids or nucleotide sequences were aligned with Muscle (Edgar 2004) and MEGA7 (Kumar et al. 2018) to obtain an optimal model that was then used to build a maximum likelihood phylogenetic tree. As an outgroup, we chose walleye dermal sarcoma virus because it was a representative of epsilonretroviruses with available full genomes that is most closely related to gammaretroviruses, the group to which REV belongs (Hayward et al. 2015). All trees were assembled with nucleotides or amino acids with MEGA7 using 1,000 bootstraps, with otherwise default values specified by the models.

Virus isolation

Virus isolation attempts were done in avian fibroblasts as described previously (Fadly et al. 2008).

Ethics

All procedures on live birds (skin biopsies) were done in accordance with the US Geological Survey, National Wildlife Health Institutional Animal Care and Use Committee number ST090715. Necropsies and tissue procurement were done on birds found dead naturally, so Animal Care and Use Committee review and approval does not apply.

Archived tissues (heart, liver, kidney, spleen, skeletal muscle, tumors) from 18 birds (1 Laysan Albatross, 17 Hawaiian Geese) originating from the Kauai (n=16) and Hawaii (n=2) islands (Hawaii, USA) collected between 1998 and 2017 were tested for REV by IHC. Three birds from Kauai, including a Hawaiian Goose with disseminated histiocytoma collected in 2016, a Hawaiian Goose with toxoplasmosis collected in 2014, and a Laysan Albatross chick with avian pox collected in 2017, tested positive by IHC (Fig. 1). Of the 18 birds tested by IHC, frozen organs were available for testing by PCR, and only the three IHC-positive birds tested positive for the REV Env gene. An additional 21 birds from the Hawaiian Archipelago, comprising 11 Laysan Albatross collected from Midway (n=5), Oahu (n=5), and Kauai (n=1), a Black-footed Albatross (Phoebastrea nigripes) from Oahu (n=1), and Hawaiian Geese from Kauai (n=7), Maui (n=1), and Hawaii (n=1) tested negative for the REV Env gene by PCR. Of 39 birds tested for REV by PCR, 23 had possibly immunosuppressive conditions including pox (n=12), inflammation (n=4), fungal infections (n=2), neoplasia (n=2), toxoplasmosis (n=2), and bacterial infection (n=1). The remainder were nonimmunosuppressive and included undetermined (n=11), emaciation (n=3), botulism type C (n=1), or trauma (n=1). There was no significant association between suspect immunosuppressive conditions and presence of REV by PCR (χ2=1.47; P=0.227).

Figure 1

Tissues from Hawaiian Geese (Branta sandvicensis; A–D) or Laysan Albatross (Phoebastria immutabilis; E and F) stained with H&E (A, C, E) or incubated with anti-reticuloendotheliosis virus (REV) antibodies and counterstained with hematoxylin (B, D, F; see Materials and Methods for details). (A) Liver with diffuse hemorrhage and necrosis and tachyzoites (arrow). (B) Same as in (A); note antibody reactivity (red) to REV in mononuclear cell cytoplasm (arrow). (C) Spleen with prominent infiltrates of histiocytes some with large pleomorphic nuclei and prominent nucleoli (arrow). (D) Same as in (C); note intracytoplasmic and nuclear staining. (E) Liver with infiltrates of macrophages and mononuclear cells (arrow). (F) Same as in (E); note staining in cytoplasm (arrow).

Figure 1

Tissues from Hawaiian Geese (Branta sandvicensis; A–D) or Laysan Albatross (Phoebastria immutabilis; E and F) stained with H&E (A, C, E) or incubated with anti-reticuloendotheliosis virus (REV) antibodies and counterstained with hematoxylin (B, D, F; see Materials and Methods for details). (A) Liver with diffuse hemorrhage and necrosis and tachyzoites (arrow). (B) Same as in (A); note antibody reactivity (red) to REV in mononuclear cell cytoplasm (arrow). (C) Spleen with prominent infiltrates of histiocytes some with large pleomorphic nuclei and prominent nucleoli (arrow). (D) Same as in (C); note intracytoplasmic and nuclear staining. (E) Liver with infiltrates of macrophages and mononuclear cells (arrow). (F) Same as in (E); note staining in cytoplasm (arrow).

Close modal

Six isolates of Avipox originating from two Apapane (Himatione sanguinea), and one each of Amakihi (Chlorodrepanis virens), House Finch (Haemorhous mexicanus), and Laysan Albatross collected between 1998 and 2004 from Hawaii or Oahu, tested negative for REV by PCR.

Virus isolation attempts on the REV-positive Hawaiian Goose with histiocytoma yielded REV, but not from the Laysan Albatross. Virus isolation was not attempted from the other REV-positive Hawaiian Goose because of insufficient sample availability.

The complete Gag, Pol, and Env genes were sequenced from the three REV-positive birds. A virus variant was classified as one with a unique concatenation of nucleotides for the Gag, Pol, and Env genes. Isoforms refer to different nucleotide sequences for a given gene. One virus variant was seen in the Hawaiian Goose with Toxoplasma gondii, four variants were seen in the Hawaiian Goose with reticuloendotheliosis, and two variants were seen in the Laysan Albatross with pox. At the gene level, two, five, and four isoforms of the Gag, Pol, and Env gene, respectively, were seen. Isoforms at the amino acid level mirrored those of nucleotides except for the Pol gene for the Hawaiian Goose with disseminated histiocytoma wherein a single isoform was seen (Table 1). Maximum likelihood trees clustered the Gag gene into three groups, with those from Hawaii birds clustering with those found in Anseriformes from China, US, and Taiwan, and in mongoose (Galidia sp.; Fig. 2). The Pol tree formed three groups, with Hawaii birds all clustering into a well-supported separate clade (Fig. 3). The Env tree formed six groups, with Hawaii birds clustering with multiple different orders and countries (Fig. 4). Trees made with amino acid sequences showed groupings similar to those seen for nucleotides for the Gag (Supplementary Material Fig. S1), Pol (Supplementary Material Fig. S2), and Env (Supplementary Material Fig. S3) genes. Sequences were deposited in GenBank with the identification numbers listed in Table 1.

Figure 2

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Gag gene. The tree was built based on the Kimura two-parameter model (Kimura 1980) with 1,000 bootstraps and rooted with walleye dermal sarcoma virus (WDSV). Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI]), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: AUS = Australia; BRA = Brazil; CHN = China; ITA = Italy; MDG = Madagascar; TWN = Taiwan; USA = United States of America. Animal group abbreviations are as follows: ANS = Anseriformes; GAL = Galliformes; MAM = Mammalia; PRO = Procelariiformes; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis). Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Figure 2

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Gag gene. The tree was built based on the Kimura two-parameter model (Kimura 1980) with 1,000 bootstraps and rooted with walleye dermal sarcoma virus (WDSV). Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI]), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: AUS = Australia; BRA = Brazil; CHN = China; ITA = Italy; MDG = Madagascar; TWN = Taiwan; USA = United States of America. Animal group abbreviations are as follows: ANS = Anseriformes; GAL = Galliformes; MAM = Mammalia; PRO = Procelariiformes; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis). Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Close modal
Figure 3

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Pol gene. The tree was built based on the Whelan and Goldman model (Wheelan and Goldman 2004) with 1,000 bootstraps and rooted with walleye dermal sarcoma virus (WDSV). Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI], USA), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: CHN = China; TWN = Taiwan; USA = United States of America. Animal group abbreviations are as follows: ANS = Anseriformes; CO = Columbiformes; GAL = Galliformes; MAM = Mammalia; PRO = Procelariiformes; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis), both from Hawaii. Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Figure 3

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Pol gene. The tree was built based on the Whelan and Goldman model (Wheelan and Goldman 2004) with 1,000 bootstraps and rooted with walleye dermal sarcoma virus (WDSV). Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI], USA), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: CHN = China; TWN = Taiwan; USA = United States of America. Animal group abbreviations are as follows: ANS = Anseriformes; CO = Columbiformes; GAL = Galliformes; MAM = Mammalia; PRO = Procelariiformes; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis), both from Hawaii. Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Close modal
Figure 4

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Env gene. The tree was built based on the Kimura two-parameter model (Kimura 1980) with gamma distribution with 1,000 bootstraps and rooted with walleye dermal sarcoma virus. Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI], USA), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: CHN = China; TWN = Taiwan; USA = United States of America. Abbreviations for animal groups are as follows: ANS = Anseriformes; COL = Columbiformes; GAL = Galliformes; MAM = Mammalia; PAS = Passeriformes; PEL = Pelecaniformes; PRO = Procelariiformes; UNK = unknown; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis), both from Hawaii. Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Figure 4

Maximum likelihood tree from nucleotide sequences of reticuloendotheliosis virus Env gene. The tree was built based on the Kimura two-parameter model (Kimura 1980) with gamma distribution with 1,000 bootstraps and rooted with walleye dermal sarcoma virus. Labels are GenBank accession, three-letter ISO3166 country code (except for Hawaii [HI], USA), and animal group. Hawaii labels end in V (virus variant) and i (gene isoform). Country abbreviations are as follows: CHN = China; TWN = Taiwan; USA = United States of America. Abbreviations for animal groups are as follows: ANS = Anseriformes; COL = Columbiformes; GAL = Galliformes; MAM = Mammalia; PAS = Passeriformes; PEL = Pelecaniformes; PRO = Procelariiformes; UNK = unknown; VAC = vaccine origin. Circles represent Hawaiian Goose (Branta sandvicensis) with neoplasia (open) or toxoplasmosis (filled), and triangles represent Laysan Albatross (Phoebastria immutabilis), both from Hawaii. Scale is nucleotide substitutions per site; numbers at nodes are bootstrap values.

Close modal

The findings of this study are significant because REV is only the second virus discovered infecting wild birds in Hawaii. The only other virus circulating in Hawaiian birds is Avipox, which has severe population effects on native Hawaiian honeycreepers (van Riper et al. 2002) and Laysan Albatross chicks (VanderWerf and Young 2016), but rarely affects Hawaiian Geese (Work et al. 2015). The paucity of viruses affecting wild birds in Hawaii, compared with the variety in North America (Fitzgerald 2007; Hansen and Gough 2007; Hollmén and Docherty 2007; Kaleta and Docherty 2007; Leighton and Heckert 2007; McLean and Ubico 2007; Paré and Robert 2007; Phalen 2007; Stallknecht et al. 2007), the nearest landmass to Hawaii, would fit the paradigm that animals colonizing isolated island ecosystems leave their parasites behind, a phenomenon known as enemy release hypothesis (ERH; Torchin and Mitchell 2004). This hypothesis postulates that parasite and pathogen communities on islands are depauperate because only the fittest animals have the ability to survive the long migrations necessary to colonize islands, and most parasites or pathogens that they harbor would likely not survive in the new environment because of lack of intermediate or susceptible hosts or vectors necessary to sustain them (Torchin and Mitchell 2004). Evidence presented for the ERH generally depends on comparison of parasite communities of hosts in their native versus introduced range (Torchin et al. 2003). Nene radiated across the main Hawaiian islands about 0.5 million yr ago from an ancestral migration of Canada Geese (Branta canadensis) to Hawaii ca. 0.9 million yr ago (Paxinos et al. 2002). More viruses circulate in Canada Geese in North America including Newcastle (Palmer and Trainer 1970), influenza (Winkler et al. 1972), goose hepatitis virus (Schettler 1971), avian pox (Cox 1980), and avian pneumovirus (Bennett et al. 2002), thereby lending credence to the ERH.

Our study is the only one we are aware of where REV has been detected in a pelagic seabird; however, few investigations of REV in seabirds exist. We found one survey of pox virus in shearwaters (Ardena spp.) from Eastern Australia that screened negative for REV by PCR (Sarker et al. 2017). Although we suspected that REV might have infected the Laysan Albatross in this report as a provirus in Avipoxvirus (Nair et al. 2013), we saw no evidence of this after a PCR screening of pox virus isolates from that case and other cases of pox in albatross and passerine birds in Hawaii. The low likelihood of REV circulating in avian pox in Hawaii would accord with findings of Kim and Tripathy (2006), who also failed to find REV in a pox isolate from a Hawaiian Goose. This paucity of retroviruses in Avipox in Hawaii contrasts with their near ubiquitous presence in poultry pox (Davidson et al. 2008). Our understanding of the drivers of REV integration into pox are rudimentary, but this phenomenon has been occurring since 1949 or earlier (Kim and Tripathy 2001). Moreover, although REV is present in Hawaii and can occasionally be associated with disease in native birds, its importance as a cause of disease seems limited based on its low prevalence in native Hawaiian birds. Surveys for REV have not been done in poultry in Hawaii, and there is no documentation of vaccination for Marek's disease or fowl pox that could explain its introduction in Hawaii (Hugh et al. 1986). According to one study, REV is a mammalian retrovirus that jumped into a malaria parasite of a pheasant at a zoo in the mid-1930s and became adapted to poultry, eventually integrating into fowl pox and gallid herpesvirus 2 (Marek's disease) via vaccine contamination (Niewiadomska and Gifford 2013). Thus, given its recent emergence in poultry, perhaps the virus is just starting to be established in Hawaii, and continued vigilance for monitoring its presence in terrestrial and pelagic seabirds is warranted. Studies on the mainland US have found low prevalence of REV in wild birds (Ferro et al. 2017; Stewart et al. 2019), according with the low prevalence seen in this study.

We saw no association between presence of REV and geese that died of conditions suggesting immunocompromise, versus other causes of death. Although REV can cause immunosuppression in birds (Nair et al. 2013), we saw no evidence of this based on pathology. Confirming this would require applying antemortem assays to assess cell and humoral immune response and associating this with infection and development of pathology. The virus can also cause tumors in birds, but we saw no evidence of association between tumors and presence of REV; however, the low sample size of birds with tumors in this study makes this conclusion tentative. In some avian taxa, such as Galliformes, tumors can be common and are often caused by viruses (Crespo et al. 2018). The endangered Attwater's Prairie Chicken is a good example where REV has posed a significant threat to birds held in captivity (Drew et al. 1998). In domestic backyard poultry, neoplasia is a leading cause of death in the US including Hawaii (Cadmus et al. 2019). By contrast, in other taxa such as Anseriformes (Fenton et al. 2018), tumors are uncommon. Future efforts to continue screening cases of neoplasia in Hawaiian birds for REV would be advisable, to see whether there is a relationship between REV infection and neoplasia. It is unlikely our IHC-positive birds were false positives, because the negative controls performed as expected (no immunoreactivity), and the staining pattern matched that reported in the literature for these reagents (Santos et al. 2008).

The significance of REV sequence variation observed herein has several possible explanations. Retroviruses as a group are known for their high degree of genetic variation, with a mutation rate close to the maximum for the genome size (Overbaugh and Bangham 2001). This variation is mainly ascribed to the poor fidelity of RNA polymerase 1, with its absence of 3′–>5′ exonuclease activity making for error-prone replication (Temin 1993). As a result, within a host, retroviruses can coexist as genetically variable quasispecies or swarms (Mansky 1998), which could explain the variation seen herein. Interestingly, the highest number of variants was seen in the Hawaiian Goose with tumors, suggesting a combination of high cell turnover and high replication rate in that bird, phenomena invoked to explain the presence of multiple virus variants in other retroviral infections such as human T-cell lymphotropicvirus 1 (Overbaugh and Bangham 2001). We saw no other differences in the virus from the Hawaiian Goose with disseminated histiocytoma that could explain its association with tumors, and alignments of all virus sequences found herein failed to match the v-rel (Stephens et al. 1983) oncogene (GenBank no. X02759.1) responsible for tumor formation for some REV viruses. Phylogeny of individual genes also differed, with the Gag and Env genes clustering with REV sequences originating from various birds and mammals from Asia, Madagascar, and North America (Fig. 2). The Pol gene formed a unique well-supported clade comprising only birds from Hawaii. This suggests that the Pol gene is under strong selective pressure, probably due to combinations of host immune response and viral replication error (Preston and Dougherty 1996), and would accord with studies of other retroviruses. For instance, in patients with human immunodeficiency virus given antiretroviral drugs, the Pol gene undergoes strong selective pressure and is important in maintaining viral fitness (Weber et al. 2003). The similar tree topology seen with nucleotides (Figs. 24) and amino acids (Figs. S1S3) lends credence to the phylogenetic relationships seen herein.

The presence of REV on Kauai only is probably a sampling artifact, in that most birds tested for the virus originated from that island. The reservoir of REV infections in Hawaii remains unknown, although backyard poultry would be logical candidates (Nair et al. 2013) given their propensity to develop tumors (Cadmus et al. 2019). Kauai has high populations of feral chickens because it is one of the few main Hawaiian islands free of Javan mongoose (Herpestes javanicus), a known predator of chickens (Duffy et al. 2015). House flies (Musca domestica) have also been documented to harbor REV, but only for short periods (<72 h), suggesting that they probably play a more limited role in transmission. Mosquitoes can also transmit the virus (Motha et al. 1984). Future studies might consider screening poultry or insect populations in Hawaii by serology or PCR, respectively, to see where the virus resides and how it might be transmitted.

Thanks to staff from Pacific Rim Conservation for access to albatross with avian pox. The use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. Sue Jarvi, James Breeden, and Mark Drew provided constructive comments on earlier versions of this manuscript. Carter Atkinson and Sue Jarvi kindly provided the pox isolates. Data for this article are available at https://doi.org/10.5066/P91MNZ32.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00164.

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Supplementary data