Conservation Genetics

, Volume 9, Issue 2, pp 339–348

Diversity, origins and virulence of Avipoxviruses in Hawaiian Forest Birds

Authors

    • Department of BiologyUniversity of Hawai‘i – Hilo
  • Dennis Triglia
    • Hawai‘i Cooperative Studies Unit, Pacific Aquaculture & Coastal Resources CenterUniversity of Hawai‘i
    • U.S. Geological Survey – Pacific Islands Ecosystems Research Center
  • Alexis Giannoulis
    • Department of BiologyUniversity of Hawai‘i – Hilo
  • Margaret Farias
    • Department of BiologyUniversity of Hawai‘i – Hilo
  • Kiara Bianchi
    • Department of BiologyUniversity of Hawai‘i – Hilo
  • Carter T. Atkinson
    • U.S. Geological Survey – Pacific Islands Ecosystems Research Center
Research Article

DOI: 10.1007/s10592-007-9346-7

Cite this article as:
Jarvi, S.I., Triglia, D., Giannoulis, A. et al. Conserv Genet (2008) 9: 339. doi:10.1007/s10592-007-9346-7

Abstract

We cultured avian pox (Avipoxvirus spp.) from lesions collected on Hawai‘i, Maui, Moloka‘i, and ‘Oahu in the Hawaiian Islands from 15 native or non-native birds representing three avian orders. Phylogenetic analysis of a 538 bp fragment of the gene encoding the virus 4b core polypeptide revealed two distinct variant clusters, with sequences from chickens (fowlpox) forming a third distinct basal cluster. Pox isolates from one of these two clusters appear closely related to canarypox and other passerine pox viruses, while the second appears more specific to Hawai‘i. There was no evidence that birds were infected simultaneously with multiple pox virus variants based on evaluation of multiples clones from four individuals. No obvious temporal or geographic associations were observed and strict host specificity was not apparent among the 4b-defined field isolates. We amplified a 116 bp 4b core protein gene fragment from an ‘Elepaio (Chasiempis sandwichensis) collected in 1900 on Hawai‘i Island that clustered closely with the second of the two variants, suggesting that this variant has been in Hawai‘i for at least 100 years. The high variation detected between the three 4b clusters provides evidence for multiple, likely independent introductions, and does not support the hypothesis of infection of native species through introduction of infected fowl. Preliminary experimental infections in native Hawai‘i ‘Amakihi (Hemignathus virens) suggest that the 4b-defined variants may be biologically distinct, with one variant appearing more virulent. These pox viruses may interact with avian malaria (Plasmodium relictum), another introduced pathogen in Hawaiian forest bird populations, through modulation of host immune responses.

Keywords

Avipox virusHawaiian birds4b core proteinA3L geneAvian pox

Introduction

The introduction of the mosquito vector, Culex quinquefasciatus, and two avian pathogens, Plasmodium relictum (avian malaria) and Avipoxvirus (avian pox) to the Hawaiian Islands is widely considered to be a primary contributing factor to population declines, extinctions and changes in the geographic and altitudinal distribution of native forest bird species in the Hawaiian archipelago (Warner 1968; van Riper et al. 1986; Atkinson et al. 1995, 2000; Woodworth et al. 2005). The mosquito vector arrived in the Hawaiian Islands around 1826 and is considered a relatively recent introduction (Hardy 1960). Both diseases are thought to have been introduced within the past 150 years to Hawai‘i, but precise dates and sites of introduction are unknown (Warner 1968; van Riper et al. 1986). Although early naturalists described pox-like lesions on dead and dying native forest birds in the 1890’s, the first definitive diagnosis for pox infection in native forest birds was not made until 1902 from an ‘Ākepa (Loxops coccineus) that was submitted to Bureau of Animal Industry diagnosticians (Henshaw 1902). The virus was diagnosed a year earlier from domestic chicken flocks on O’ahu, but no other information is available regarding its point of entry or how rapidly it spread among the main islands. Perkins (1903) reported more frequent occurrence of pox-like lesions in wetter, windward forests, an observation which supports the role of C.quinquefasciatus as the primary vector of the virus. Domestic poultry has long been assumed to be the most likely route for introduction of the virus to Hawai’i, yet recent studies suggest that isolates circulating in native forest birds are distinct from fowlpox and have a higher diversity of restriction fragment length polymorphism (RFLP) patterns than might be expected from a single introduction (Tripathy et al. 2000; Kim and Tripathy 2006).

The role that avian pox has played in the demise of the Hawaiian avifauna independent of avian malaria has been difficult to determine from field observations. Both malaria and pox occur together more frequently than expected by chance (van Riper et al. 1986; Atkinson et al. 2005), and demographic studies carried out to date have not accounted for this possible interaction (VanderWerf 2001). Recent studies indicate that through mixed infections, even avirulent pathogens can play a significant role in influencing the outcome of coupled host-pathogen systems (Thomas et al. 2003). These results, considered together with theoretical predictions, suggest that mixed infections could significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies and evolution of virulence. This introduced avian disease system in Hawaii is an exceptional natural system in which to investigate mixed infections involving multi-host pathogens and evolution of virulence. In this study, we describe host range, geographic range and genetic diversity of field isolates of avian pox from four main Hawaiian Islands. We also provide genetic documentation of one isolate from a museum specimen from the beginning of the 20th century. Finally, we provide preliminary evidence for possible differences in virulence of two genetically distinct isolates in experimentally infected Hawai‘i ‘Amakihi (Hemignathus virens).

Methods

Collection of modern pox lesions

Pox-like lesions were collected from the feet and legs of birds that were found dead in the field or handled during routine mist-netting operations. Lesions were collected from a variety of host species and locations on the islands of Hawai‘i, Maui, Moloka‘i and O‘ahu between 1993 and 2005, with most collections on Hawai‘i Island between 2001 and 2005 (Table 1). Lesions from dead birds were excised with a sterile scalpel and placed in sterile plastic bags; scabs from lesions on live birds were collected by flaking superficial portions of the lesions into dry, sterile vials. All samples were stored frozen at −20°C for up to 12 years before virus isolations were attempted.
Table 1

Isolate designation, host order, species, geographic origin and year of sample collection for pox isolates used in this study

Isolate

Host Order

Host Species

Island

Locality

Date

Chicken 23

Galliformes

Gallus domesticus

Hawai‘i

Ola‘a Forest

1993

Chicken 1

 

Gallus domesticus

Hawai‘i

Volcano

2001

House Finch 24

Passeriformes

Carpodacus mexicanus

Hawai‘i

Pu‘u La‘au

1998

Hawai‘i ‘Amakihi 22

 

Hemignathus virens

Hawai‘i

Volcano

1998

‘I‘iwi 2

 

Vestiaria coccinea

Hawai‘i

Hakalau

2001

‘Āpapane 5

 

Himatione sanguinea

Hawai‘i

Volcano

2002

Palila 17

 

Loxioides bailleui

Hawai‘i

Volcano

2002

‘Alalā 21

 

Corvus hawaiiensis

Hawai‘i

Volcano

2003

Hawai‘i ‘Amakihi 15

 

Hemignathus virens

Hawai‘i

‘Ainahou

2003

‘Āpapane 16

 

Himatione sanguinea

Hawai‘i

Keauhou

2003

Hawai‘i ‘Amakihi 18

 

Hemignathus virens

Hawai‘i

Kaupulehu

2004

Hawai‘i ‘Amakihi 27

 

Hemignathus virens

Hawai‘i

Brysons

2005

House Finch 4

 

Carpodacus mexicanus

Maui

Wailuku

2002

‘Āpapane 14

 

Himatione sanguinea

Moloka‘i

Kamakou

2003

Laysan Albatross 19

Procellariformes

Diomedea immutabilis

O‘ahu

Ka‘ena Point

2004

Maintenance of cell cultures

Cryopreserved Muscovy duck (Cairina moschata) embryonic fibroblasts (MSDEF) were generously provided by Dr. Wallace R. Hansen of the National Wildlife Health Center in Madison, WI. The MSDEF were propagated in Medium 199 (GIBCO #11150-059, Grand Island, New York, USA) with Earle’s salts and 2.2 g/l sodium bicarbonate supplemented with 10% Fetal Bovine Serum (GIBCO #16000-044; certified origin: USA), 100 μM MEM Nonessential amino acids (GIBCO #11140-050), 2 mM l-glutamine (GIBCO #25030-081), MEM Vitamin Solution (GIBCO #11120-052) and 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate and 250 ng/ml amphotericin B (as Fungizone) (GIBCO #15240-062). Fibroblasts were grown in a 5% CO2/95% humidified air incubator at 37°C to confluence in T-75 vented tissue culture flasks (Corning) and were split 1:4 by trypsinization (0.05% Trypsin; 0.53 mM EDTA 4Na; GIBCO #25300-054) every 7 days.

Virus isolation and propagation

Individual pox lesions obtained from 15 birds were processed separately using the following procedure. A lesion was ground for 2 min using a 5 ml Wheaton Potter-Elvehjem tissue grinder containing 4.5 ml Hank’s Balanced Salt Solution (HBSS; GIBCO #24020-117) supplemented with 5% (v/v) glycerol (Sigma Chemical Company #G-2025, St. Louis, Missouri, USA), 300 U/ml penicillin G sodium, 300 μg/ml streptomycin sulfate and 750 ng/ml amphotericin B (as Fungizone) (GIBCO #15240-062). After grinding, the homogenate was transferred to a 15 ml polypropylene conical centrifuge tube (Corning) and centrifuged for 30 min at 800 × g at room temperature. About 5 ml of the supplemented HBSS was also centrifuged for subsequent use as a “sham” infection inoculum. After the centrifugation, the supernatant fluids (pox and sham) were carefully transferred to new sterile centrifuge tubes and refrigerated at 2–8°C for 2 h prior to inoculation of the MSDEF monolayer cultures. Two and one-half milliliters of the clarified pox inoculum and 2.5 ml of the HBSS “sham” inoculum were each added into a 7–day old confluent T-75 flask of MSDEF containing 22.5 ml of completely-supplemented Medium 199 and the flasks were returned to the 37°C, 5% CO2 humidified air incubator. The medium was changed 3 and 5 days post inoculation. Seven days post-inoculation, the T-75 flasks were freeze-thawed and then the cells were manually scraped off the flask surface. To complete the lysis of the host cells, the lysate and sham control medium were vortexed vigorously in sterile 50 ml Falcontm conical centrifuge tubes for 2 min each. The tubes were then centrifuged for 30 min at 800 × g, the supernatants transferred to fresh tubes which were refrigerated for 2 h at 2–8°C, then passaged into fresh confluent T-75 flasks of MSDEF cells. No prior passage onto a chorioallantoic membrane (CAM) of a chicken embryo was necessary, as cytopathic effect (CPE) were apparent in the pox-infected MSDEF culture, but not in the sham-infected culture.

Preparation of inocula for experimental infections

Seven days post-infection, pox-infected and sham-“infected” Muscovy duck embryonic fibroblast monolayer cultures in T-75 flasks were incubated for 30 min at 37°C/7.5% CO2 with Hank’s Balanced Salt Solution (HBSS; GIBCO #24020-117) to reduce the amount of residual fetal bovine proteins from the tissue culture medium. The HBSS was decanted to waste and 1.5 ml of sterile-filtered Phosphate Buffered Saline, pH 7.4 (PBS; prepared in our laboratory) was added to each monolayer. The cells were then lysed by freeze-thawing the entire flask(s), followed by manually scraping the adherent host cells off the flask surface with sterile cell scrapers (Fisher Scientific) and collecting the lysates into sterile 15 ml Corningtm conical centrifuge tubes. The pox and sham control lysates were vortexed vigorously and continuously for 2 min each, then centrifuged for 15 min at 800 × g to remove host cell debris. All viral and sham material used in this experiment had been passaged a minimum of 3 times (maximum of 5 times) in vitro.

DNA Extractions and PCR amplifications

DNA was extracted from 2 ml pox culture lysates using the DNeasy Tissue Kit (Qiagen) following modified (scaled up) manufacturer’s protocols. Between 4 and 10 μl of extracted DNA were used in 25 μl PCR reactions containing 1× reaction buffer (Promega), 0.8 mM of dNTP, 1.25 units of Taq polymerase (Promega), 2 mM MgCl2 and 0.8 μM of each primer. Primers used were P1 and P2 (Lee and Lee 1997), with an expected approximate 580 bp amplification product originating from the gene coding for the 4b core protein. Samples were subjected to an initial denaturation step of 4 min at 94°C followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 53°C, extension for 1 min at 72°C in a MJ Research PTC-100 thermocycler using the hot lid. Products from the PCR were visualized by electrophoresis on a 2% agarose gel.

Cloning and sequencing

PCR products were cloned using the TOPO-TA Cloning Kit (Invitrogen) following the manufacturer’s protocols. Colonies were screened for insert size by PCR with plasmid based M13 primers. Plasmids were isolated using the QIAprep Spin Miniprep Kit Protocol (Qiagen) following the manufacturer’s protocol. Selected clones were sequenced in both directions on a Beckman CEQ 8000 (Core Genetics Facility, University of Hawai‘i at Hilo) using modified M13 primers (Jarvi and Farias 2006). All sequences were proofed and analyzed using Sequencher® (GeneCodes Corp). Sequences presented here have been deposited in GenBank (EF568377-EF568404).

Museum samples

Tissue samples from eight ‘Elepaio (Chasiempis sandwichensis) museum skins with obvious pox-like lesions were kindly provided by the Bernice Pauahi Bishop Museum, Honolulu, Hawai‘i. All specimens dated from between 1900 and 1903; four were collected on the island of O‘ahu and four on the island of Hawai‘i. Tissue scrapings were collected from lesion sites, placed in DNA lysis buffer (0.1 M Tris–HCl pH 8.0, 0.1 M sodium EDTA, 2% SDS) and transported on ice before storing at −20°C. All subsequent work with these samples was carried out in a separate laboratory designated for museum tissue work. DNA was extracted using the Qiagen DNeasy Tissue Kit and standard protocols, except that samples were eluted twice in 100 μl buffer AE, with elutions kept separate to prevent dilution. Samples were extracted in batches of five or less, and each batch included an extraction blank to monitor for cross contamination. Extraction blanks were carried through subsequent PCR steps. For PCR, 14 μl of genomic DNA were used as the template in a 25 μl PCR with primers PV4B.P4 (5′-GCACATGTTAAGGGGTCTCTATC-3′) and PV4B.P5 (5′-TGTAGTATCAATAAGCGCTTGGT-3′) at 0.8 μM each. These primers were expected to amplify a 116 bp product from the 4b core protein gene; the shorter fragment length was chosen to maximize the chances of obtaining PCR product from potentially degraded template DNA. All other conditions were as described above for primer set P1/P2, except that the number of cycles was increased from 40 to 55. PCR reactions, including a negative control, were prepared in the museum lab and transported on ice to the main lab, where 1 μl of genomic DNA from a modern isolate (‘Amakihi B07) was added to the positive control reaction prior to thermocycling. To confirm results, a complete second PCR trial was performed using the same samples and conditions, but with a second genetically distinct positive control (‘Āpapane 16.2). PCR products were visualized as described above. All visible bands from museum samples, along with the positive controls, were cloned directly from the PCR using the TOPO-TA cloning kit (Invitrogen) and following the manufacturer’s protocols, except that the ligation time was increased from 15 min to between 75 and 90 min to increase cloning efficiency. Cloning of products from the first and repeat PCR was conducted separately and the negative control reaction from the repeat PCR was also cloned to monitor for low levels of contamination. Colonies were screened for insert size as above. Overnight cultures were started from randomly selected successful transformants, and plasmid DNA was collected using the QIAprep Minispin Kit (Qiagen). Four plasmids from each museum cloning reaction and two plasmids from each positive control were sequenced as above.

Phylogenetic and diversity analyses

We assessed the relationship of the sequences by constructing a neighbor-joining tree (Saitou and Nei 1987) and calculated nonsynonymous (dN) and synonymous (dN) substitutions and cluster diversity from Kimura 2-Parameter corrected distance matrices using MEGA 2.0 (Kumar et al. 2000). In addition to the sequences obtained in this study, we included eleven other sequences for phylogenetic comparison (see legend, Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10592-007-9346-7/MediaObjects/10592_2007_9346_Fig1_HTML.gif
Fig. 1

A neighbor-joining tree (Kimura 2-parameter corrected distances) generated using MEGA (Kumar et al. 2000). Bootstrap values (NJ/ME) are indicated at nodes (1000 replications). Other 538 bp sequences included for comparison are AY318871 ATCC Canary, AY30309 Canary, AY30308 Sparrow, AY530303 Pigeon, AY30310 Curlew, AY530304 Turkey, AY530305 Ostrich, AY530311 Love bird and Fowlpox sequences M25781, AY530302, and AJ005164. Abbreviations are as follows for species: HAAM (Hawai‘i ‘Amakihi), APAP (‘Āpapane), ALAL (‘Alalā), PALI (Palila), LAAL (Laysan albatross), HSFN (House finch), CNRY (Canary) and CHKN (Chicken). Numerical isolate designations are followed by island of origin abbreviation (HI, Hawai‘i; MA, Maui; MO, Moloka‘i; OA, O‘ahu) and year of sample collection. Insert: A similarly generated tree of sequences obtained from five clones from one ‘Amakihi (HAAM 22), one ‘Āpapane (APAP 5), and from two clones each from one House Finch (HSFN 24) and one Chicken (CHKN 23). The sequences originating from the isolates used in experimental infections are indicated by * (Cluster 1) and ** (Cluster 2)

Experimental infections

We conducted experimental infections (University of Hawai‘i Institutional Animal Care and Use Committee Protocol 00-035) with isolates from two pox virus variants (isolate 1 from ‘Amakihi 22 and isolate 2 from ‘Amakihi 15 in Fig. 1) from the island of Hawai‘i to collect preliminary data about their relative virulence in a native species and assess host range in two common non-native passerines, House Sparrow (Passer domesticus) and Japanese White Eye (Zosterops japonicus). Four Hawai‘i ‘Amakihi from previous experimental studies of avian malaria were used for the pox experiment. Birds were adapted to captivity in a vector-proof aviary at Hawai‘i Volcanoes National Park as described previously (Atkinson et al. 2000) and screened for antibodies to pox virus using a modification of the ELISA protocol described by Kim and Tripathy (2006). Three birds were collected at xeric, high elevation (2000 m) habitats on Mauna Kea Volcano where prevalence of pox and malaria is extremely low (‘Amakihi #167, #11939 and #11941), while one ‘Amakihi was captured at low elevation (50 m) wet forest at Malama Ki Forest Reserve on the southeastern slope of Kilauea Volcano (‘Amakihi #41538). Two of the three high elevation ‘Amakihi and the single low elevation bird had recovered from acute malarial infections over one year prior to exposure to pox virus while the third high elevation ‘Amakihi was an uninfected control bird from a prior malaria experiment. None of the birds had serological evidence of prior exposure to pox virus.

Two ‘Amakihi (#167 and #41358) were infected with isolate 1 (‘Amakihi 22) by needle pricks in the foot pad with a virus suspension derived from cultured MSDEF both to mimic mosquito feeding and because lesions from wild birds are most often found in this area (C.T. Atkinson, unpublished data). A sterile 27-gauge syringe needle was dipped in the virus suspension and used to lightly prick the foot pad. This was repeated 4 times for each bird. As controls, two sham-infected Hawai‘i ‘Amakihi (#11939 and #11941) were pricked five times in the foot pad with a sterile 27-gauge syringe needle that was dipped into a virus free (“sham”) suspension of cultured MSDEF and housed in a separate room to avoid cross contamination with the virus. Lesion development was monitored by photography until lesions resolved.

At day 251 after initial exposure to avipox isolate 1, ‘Amakihi #167 and ‘Amakihi #41538 were rechallenged by needle pricks in the foot pad with pox isolate 2 (from ‘Amakihi 15). The two previously sham-inoculated ‘Amakihi (#11939 and 11941) were similarly exposed to isolate 2 at the same time to serve as positive controls. Concurrently, two House Sparrows and two Japanese White Eyes were exposed to pox isolate 2 by needle pricks to the foot pad and two additional House Sparrows and two additional Japanese White Eyes were inoculated with a sham suspension to serve as uninfected controls in this experiment. In both experiments, sham-infected birds were maintained in a separate room to avoid cross contamination. All birds were monitored for 2 months to assess lesion development after exposure to pox isolate 2. All birds that died during the course of the experiment were necropsied and representative pieces of lesions and major organs were fixed in buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Results

Fifteen pox virus isolates were prepared in Muscovy duck cell lines from lesions collected between 1993 and 2005 from eight species of birds (Table 1). Isolates included for genetic characterization originated from Moloka‘i (‘Āpapane, Himationesanguinea), O‘ahu (Laysan Albatross, Diomedea immutabilis), Maui (House Finch, Carpodacus mexicanus) and various species on Hawai‘i Island including domestic chickens (Gallus gallus), ‘I‘iwi (Vestiaria coccinea), ‘Āpapane, captive ‘Alalā (Corvus hawaiiensis) , Hawai‘i ‘Amakihi and captive Palila (Loxioides bailleui). Cytopathic effects (CPE), including presence of viral inclusion bodies (Bollinger bodies) and rounding and detachment of cells, were evident in cultured MSDEF within 7 days after infection with pox isolates, but not in parallel sham-infected cultures.

PCR primers specific for the 4b fowlpox gene (Lee and Lee 1997) were used successfully to amplify, clone and sequence a 538 bp gene fragment from these 15 isolates. In total, 26 sequences were obtained and these were subjected to phylogenetic analyses along with other published sequences (Fig. 1). The sequences obtained from isolates originating from seven species form two distinct clusters, with the chicken sequences forming a distinct basal cluster with other fowlpox sequences. Genetic distances within Hawaiian clusters 1 and 2 are minimal (d = 0.004 SE 0.001 and d = 0.001 SE 0.000, respectively). Mean net distance between the Hawaiian sequences forming the two variant clusters is 0.056 SE 0.011. The ratio of nonsynonymous to synonymous substitutions (dN/dS) is 0.0932 in this gene region. Both clusters contain sequences from multiple species from multiple geographic origins over similar timeframes. Five clones were sequenced from an ‘Āpapane, five from a Hawai‘i ‘Amakihi, two from a House Finch and two from a chicken to evaluate within-individual variation. Within-individual variation appears minimal (Fig. 1 inset), with 1/5 ‘Āpapane clones differing from the others by only a single nonsynonymous nucleotide substitution, and 2/5 ‘Amakihi clones differing from the others by two nucleotides (one dN, one dS). It is unclear whether these minor changes represent valid variant distinctions as the potential for error introduced by PCR cannot be disregarded. The pox variants detected in chickens cluster closely with other fowlpox sequences and appear distinct from variants detected in Hawaiian birds. The three clones originating from two individuals are very similar (d = 0.002; SE 0.002), but differ from one another by one nucleotide resulting in an amino acid substitution. Divergence of these chicken sequences from the Hawaiian sequences in variant cluster 1 is d = 0.324 (SE 0.028), and from the Hawaiian sequences in variant cluster 2 is d = 0.335 (SE 0.025).

A total of eight identical 116 bp sequences (represented by ELEP 1.1 in Fig. 2) was obtained from clones from the lesion of one individual sample BBM3846 collected 08 March 1900, from Kuaia, Hawai‘i (east Hawai‘i Island). Relationships between this museum sequence and those generated from the modern isolates (bases 294–409) were estimated by K2P distance (Bootstrap, 1000 replications) and are depicted in a neighbor-joining tree (MEGA 2.0) in Fig. 2. These sequences were obtained from clones originating from two separate PCR trials that used distinct modern isolates from the two different clusters as positive controls (‘Amakihi B.07 in Cluster 2 in one reaction, ‘Āpapane 16.2 in Cluster 1 in a second reaction). The 1900 ELEP 1.1 sequence clusters basal to the seven other sequences in cluster 2 that are identical to each other at this 116 bp region. Divergence of ELEP 1.1 from control HAAM B.07 in cluster 2 is 0.0086 and from control APAP 16.2 (in cluster 1) is 0.0776.
https://static-content.springer.com/image/art%3A10.1007%2Fs10592-007-9346-7/MediaObjects/10592_2007_9346_Fig2_HTML.gif
Fig. 2

A neighbor-joining tree (K2P) generated to estimate the relationship of the historical 116 bp ‘Elepaio 1.1 4b sequence (circa 1900) to sequences from recently collected samples (MEGA; Kumar et al. 2000). Bootstrap values (NJ) are indicated at nodes (1000 replications). Abbreviations and sequences included for comparison are as in Fig. 1

In our experimental infections, two Hawai‘i ‘Amakihi exposed to pox isolate 1 (‘Amakihi 22) (#167 and #41358) developed small circular sores at the inoculation site that were several mm in diameter by Days 32 and 39 post-infection (PI), respectively. Within 10 days after their initial appearance, lesions developed rapidly in both birds and became swollen and scabby. In ‘Amakihi #167, the scabby area spread from the foot pad to the hind toe by Day 60 PI (Fig. 3B). By Day 94 PI swelling had decreased and the lesion was completely healed by Day 103 PI. Lesion resolution was faster in ‘Amakihi #41538. Swelling was decreasing and the lesion was rapidly healing by Day 49 PI. The lesion was completely healed by Day 67 PI. Hawai‘i ‘Amakihi controls that were inoculated with the sham suspension (#11939 and #11941) did not develop lesions.
https://static-content.springer.com/image/art%3A10.1007%2Fs10592-007-9346-7/MediaObjects/10592_2007_9346_Fig3_HTML.jpg
Fig. 3

Pox lesions on foot of Hawai‘i ‘Amakihi #167, following primary infection with an isolate from variant cluster 1 (‘Amakihi 22) in the left foot pad (A, B) and rechallenge with an isolate from variant cluster 2 ( ‘Amakihi 15) in the right foot pad (C, D). (A) Pre-infection left foot (banded leg). (B) Lesions on foot pad and hind toe (arrows) from variant 1 at Day 61 post-infection. (C) Pre-infection right foot. (D) Proliferative lesion on right foot from variant 2 at Day 55 post-infection. The ‘Amakihi had recovered completely from infection with variant 1 before being rechallenged with variant 2, 251 days after the initial infection

These four birds were then challenged with isolate 2 (‘Amakihi 15). In ‘Amakihi #167, after rechallenge with isolate 2, lesions first became evident at Day 21 and expanded rapidly, becoming swollen and bloody by Day 28 PI (Fig. 3). By Day 35 PI the lesion was substantially larger and ‘Amakihi #167 had difficulty lifting its foot (Fig. 3D). By Day 43 PI, the lesion had spread to the skin around the base of the beak, and ‘Amakihi #167 succumbed on Day 49 PI. At necropsy, the beak lesions were large and interfered with vision in both eyes, but did not extend into the buccal cavity. The foot lesion was massive, bloody and involved all 4 digits. Abundant eosinophilic viral inclusion bodies (Bollinger bodies), ballooning of infected epithelial cells, and extensive proliferation of the epithelium was evident in histological sections of the lesions, providing evidence that avipox virus was present. By contrast, low elevation ‘Amakihi #41538 failed to develop lesions by Day 79 PI when the experiment was terminated.

Lesion development in the two naïve control birds (#11939 and #11941) infected with pox isolate 2 became evident at Days 43 and 28, respectively, and progressed rapidly in a manner similar to ‘Amakihi #167. The foot lesion on ‘Amakihi #11939 was massive and swollen by Day 56 PI, and had spread to the skin immediately above the tibio-tarsal joint. The foot lesion on ‘Amakihi #11941 expanded rapidly after and involved most of the foot by Day 43 PI. Both ‘Amakihi #11939 and #11941 succumbed on Days 58 and 48 PI, respectively. At necropsy, both birds had massive foot lesions that involved all 4 digits and histological sections of lesions had abundant Bollinger bodies and were similar to those described above.

By contrast to experimentally-infected Hawai‘i ‘Amakihi, no lesions developed on either pox- or sham-infected House Sparrows (n = 4) or Japanese White Eyes (n = 4).

Discussion

In vaccinia (Orthopoxvirus) the 4b polypeptide, a major virion structural protein (Moss 1978; 2001), is encoded by the A3L gene and is necessary for production of a transcriptionally active virion particle (Kato et al. 2004). The gene encoding the fowlpox virus 4b core protein has been identified by analogy to the vaccinia 4b gene (Binns et al. 1989). Phylogenetic analysis of the 4b sequences obtained in this study indicate that one of the two 4b variants detected in Hawaiian forest birds (variant 1) appears closely related to the ATCC canarypox variant (AY318871, Fig. 1). These results are remarkably similar to recent findings from Galapagos Finches (Thiel et al. 2005) which document two canarypox variants, Gal 1 and Gal 2, one of which (Gal 1) is more similar to canarypox than the other (Gal 2). The Galapagos variants are defined by sequence distinctions between the canarypox CA.X and thymidine kinase genes and the canarypox CA.2 and CA.3 genes.

Generally, avian poxviruses tend to be very host-specific, especially those infecting wild birds (Tripathy 1993). Divergence of the two variant clusters in our study was 0.056 and strict host specificity was not apparent among the 4b-defined variants originating from native birds. The variant 1 cluster is comprised of isolates originating from four distinct host species including honeycreepers (Hawai‘i ‘Amakihi and ‘Āpapane ), a crow (‘Alalā) and a seabird (Laysan albatross). The variant 2 cluster contains isolates originating from four native (Palila, ‘I‘iwi, ‘Āpapane, Hawai‘i ‘Amakihi) and one introduced passerine (House Finch). We detected no obvious temporal association as both variants were detected in Hawai‘i ‘Amakihi from Hawai‘i Island in 1998, 2004 and 2005 (variant 1) and 2003 (variant 2) and in ‘Āpapane in 2003 (variant 1) and 2002 (variant 2). No clear association with geographic origin was found based on these 4b gene fragment sequences. Isolates in the variant 1 cluster originated from multiple geographic sites in Hawai‘i (Hawai‘i Island, Moloka‘i, and O‘ahu) and isolates in the variant 2 cluster from Hawai‘i Island and Maui, over similar timeframes. The variant 1 cluster however, includes several sequences of non-Hawai‘i origin as well suggesting a higher degree of conservation (ATCC canary, other canary, curlew, sparrow), whereas variant 2 appears to be more specific to Hawai‘i.

It is known that mosquitoes can infect different species of birds after a single feeding on an animal infected with avian poxviruses (Tripathy 1993), and we found that individuals of at least two native species (‘Amakihi and ‘Āpapane) can be infected by either variant (see Fig. 1). However, the minimal variation detected among multiple clones within single individuals provides no evidence for simultaneous co-infection of any individuals with both 4b variants. Birds appear to be infected either with variant 1 or with variant 2.

Analysis of this relatively small data set suggests that both variants are likely ubiquitous throughout the archipelago and infect multiple species, but further analyses of additional genes and an expanded epidemiological study are needed to refine variant distinctions before more definitive statements can be made about their geographic distribution, host ranges and temporal occurrence.

The sequences of the genomes of over 50 members of the Family Poxviridae are currently available (see Poxvirus Bioinformatics Resource, www.poxvirus.org). Among these, the genome of the Avipoxvirus (fowlpox virus, FPV-FCV, Afonso et al. 2000) is noted as being the largest. Whereas gene order and spacing appear highly conserved in other chordopox genomes, unique gene rearrangement events have occurred in the fowlpox genome that have interspersed variable gene regions within otherwise highly conserved core regions (McLysaght et al. 2003; Lefkowitz et al. 2006). Based on molecular clock estimates of mutations accumulating in poxviruses at a rate of 0.9–1.2 × 10−6 substitutions/nucleotide site/year, Avipoxvirus diverged from mammalian viruses approximately 420,000 years ago (Babkin and Shchelkunov 2006). This mutation rate estimate was based on analyses of the conserved central region (approximately 102 kb) of the orthopoxvirus genome and the DNA polymerase gene in different genera (Babkin and Shchelkunov 2006). The AL3 gene encoding the 4b polypeptide is located within this highly conserved region (www.poxvirus.org). Since mosquitoes were introduced to the Hawaiian Islands only relatively recently (1820’s; Hardy 1960) extensive transmission of these viruses did not likely precede this approximate time period. Given the observed divergence between both 4b clusters (0.056) and from the fowlpox-like variant found in chickens (>0.324) and the low estimated mutation rate (approximately 1.0 × 10−6 ), the variation between these pox variants appears too great to have occurred due to mutation alone within the given timeframe. Also, this gene fragment does not appear to be under positive selection with a ratio of nonsynonymous to synonymous substitutions of less than one. Thus, there is a high likelihood that the existence of these three variants in the Hawai‘i is due to multiple, most probably independent introductions, and does not support the hypothesis of infection of native Hawaiian species through the introduction of infected fowl.

We present evidence that at least one of the two variant-types detected has been present in Hawai‘i for over 100 years with the amplification of a 4b pox gene sequence from an ‘Elepaio collected on the windward side of Hawai‘i Island in 1900. To obtain this sequence we completed two separate trials which involved independent PCR amplification, cloning and sequencing of clones using birds from different variant clusters as positive controls to be able to rule out any contaminating DNA originating from positive controls. We also completed many other precautionary steps (described above) to avoid the possibility of contamination, and if contamination occurred, to be able to detect it. This 1900 ‘Elepaio sequence is representative of 8 identical clones obtained from two distinct PCR/cloning trials and is unique among all sequences obtained to date. It is highly unlikely an artifact of contamination. The fact that it is most similar to isolates in the variant 2 cluster is interesting because the isolate from one individual from the variant 2 cluster (‘Amakihi 15) appears to be more virulent than the isolate evaluated from the variant 1 cluster (‘Amakihi 22) based on these preliminary studies. Comparison of present-day virulence with historical virulence would require culturing of isolates from the turn of the century, but viability of virus isolates from museum samples is currently unknown. Direct comparison with historical virulence is yet further complicated by the fact that virulence is a term that is relative to the host-parasite system within which it is defined and historical hosts are currently unavailable.

Two primary types of disease result from infection with avian poxviruses: the cutaneous and the diphtheritic forms. Clinical symptoms observed in birds infected with pox in Hawai‘i closely resemble those of the cutaneous form (van Riper et al. 2002, vanderWerf 2000, Tripathy et al. 2000, Atkinson et al. 2005). In this study, cutaneous lesions for both variant 1 and 2 isolates were slow to develop in Hawai‘i ‘Amakihi, first becoming evident between 3 and 6 weeks after infection. The variant 1 isolate produced a relatively mild, self-limiting infection and lesions were relatively small in size and did not interfere with perching. By contrast, the variant 2 isolate produced a proliferative, rapidly fatal infection. While it is not possible to draw firm conclusions about relative pathogenicity of these two variants because of small sample sizes, it is noteworthy that all birds (3/3) that developed lesions after viral challenge with variant 2 died within 50 days after infection. Lesions in all three cases were massive and disruptive of foot function. One Hawai‘i ‘Amakihi (#41358) failed to develop lesions after rechallenge, but it is difficult to determine whether this individual had residual immunity from the previous infection with variant 1 (in contrast to the other rechallenged bird) or whether it simply failed to become infected by needle prick. We were not able to infect either Japanese White Eye or House Sparrows with an isolate of variant 1, which suggests that this variant may exhibit some host specificity among avian species even though closely related isolates were recovered from hosts from two avian orders (Procellariformes and Passeriformes). Additional work is needed to better define the host range and pathogenicity of both variants.

The immune response to poxviruses involves both innate and adaptive immunity including both antibody (humoral) and cell-mediated responses (Smith and Kotwal 2002). Poxviruses possess genes that especially modify the innate mechanisms of the host immune response, therefore sustaining viral persistence during early infections. Proteins encoded by the large poxvirus genomes include genes that encode complement control protein, interleukin-1 receptor homologue, tumor necrosis factor receptor homologue, and interferon receptor homologue (Kotwal 1997; Smith and Kotwal 2002). Malaria and pox occur together more frequently than expected by chance in Hawaiian forest birds for reasons that are not entirely clear (van Riper et al. 1986; Atkinson et al. 2005). This is most probably not coincidental given the potential immunocompromising capabilities of poxviruses. If susceptible native birds are infected with poxvirus, reduced inflammatory responses and cell-mediated responses may, when co-infected with malaria, result in more rapid parasite expansion and thus increased virulence due to a coupled host-pathogen system.

The combination of a relatively recent introduction of a highly efficient mosquito vector and two avian pathogens (a protozoan and a virus) to an isolated island ecosystem with naïve, highly susceptible avian hosts provides unique opportunities to investigate evolution of virulence in a natural system. The extreme isolation of the archipelago, the mixture of endemic and exotic host species and the diversity of habitats make this island chain an exceptional natural laboratory for investigating these evolutionary processes.

Acknowledgements

This work was supported in part by National Science Foundation Grant DEB 0083944 and the USGS Wildlife and Invasive Species Programs. We thank Carla Kishinami and the Vertebrate Zoology Section at the Bernice Pauahi Bishop Museum, Honolulu, Hawaii for cooperation and assistance in collecting pox lesions from ‘Elepaio specimens in their collection, Wallace Hansen at the USGS-National Wildlife Health Center for supplying embryonic Muscovy Duck fibroblasts and advice for pox virus cultivation, Alan Lieberman at the Keauhou Bird Conservation Center for pox virus lesions from captive Palila and ‘Alalā, Eric VanderWerf, Fern Duvall, Sam Aruch, Julie Lease, Donna Ball, Eric Tweed, Paul Banko, and Colleen Cole for collecting pox lesions from other species and Leayne Patch for assistance with aviary experiments. Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the U.S. Government. Mahalo i ka po‘e o Hawai‘i no ko lākou kōkua a me ko lākou aloha. Thank you very much to the people of Hawai‘i for their help and aloha.

Copyright information

© Springer Science+Business Media B.V. 2007