Core Protein Gene Research Articles

An analysis of the molecular evolution of Hepatitis B viral genotypes A/B/D using a Bayesian evolutionary method

BackgroundHepatitis B virus (HBV) infection is a major global health problem. The infectious virion contains an inner “core particle”, which is made of 180 or 240 copies of core protein, alternatively known as hepatitis B core antigen, or HBcAg which encloses the viral genome.MethodIn this study, we characterized HBV genotypes and used Bayesian analyses to estimate date of emergence of the most recent common ancestor (TMRCA) of three HBV genotypes, A, B, and D.ResultsWe estimated that the rate of evolution of HBV core protein gene to be 1.127 (0.925–1.329, 95% HPD) substitutions per site per year. The TMRCA of HBV for genotypes A, B, D were 118 (54–194, 95% HPD) year, 184 (78–323, 95% HPD) year and 133 (65–230, 95% HPD) year, respectively. Demographic histories of the HBcAg gene showed that the relative genetic diversity had a sharp increase within the first 10 years of its emergence.ConclusionUsing a bayesian evolutionary method to predict the outbreak trends of HBV through evolutionary trees of HBV, and provide theoretical foundations for clinical prevention and treatment of HBV.

127. Therapeutic Efficacy in a Breast Cancer Model of Novel Conditionally Replicative Adenovirus (CRAd) Based upon the Deletion of Minor Core Protein Gene V

127. Therapeutic Efficacy in a Breast Cancer Model of Novel Conditionally Replicative Adenovirus (CRAd) Based upon the Deletion of Minor Core Protein Gene V

Experimental Evolution of a Bacteriophage Virus Reveals the Trajectory of Adaptation across a Fecundity/Longevity Trade-Off

Life history theory attempts to account for how organisms lead their lives, balancing the conflicting demands of reproduction and survival. Here, we track the genomic and phenotypic evolution of the bacteriophage virus T7 across a postulated fecundity/longevity constraint. We adapted T7 to a challenging survival environment (6M urea). Our evolved strain displayed a significant improvement in propagule survival, coupled with a significant loss of fecundity (reduced growth rate on host cells). However, the increased resistance to urea did not generalise to increased resistance against temperature stress, highlighting that propagule durability is environment dependent. Previous comparative studies predicted that changes in propagule resistance would be mediated by changes in capsid proteins or gene deletions. In contrast, we found that point mutations in internal core protein genes (6.7 and 16) were responsible for the increased urea resistance of our evolved strain. Prior to the emergence of the 6.7 and 16 mutations, a distinct set of 5-point mutations peaked at over 20% prevalence before attenuating, suggestive of negative epistatic interactions during adaptation. Our results illustrate that parasites can adapt to specific transmission environments, and that this adaptation can impose costs on the subsequent ability to exploit host cells, potentially constraining durable parasites to lower virulence.

Avipoxvirus infections in brown kiwi (Apteryx mantelli)

CASE HISTORY: Nodular lesions were found on the skin of two immature brown kiwi (Apteryx mantelli) less than 6 months of age living freely on Ponui Island off the North Island of New Zealand. The lesions were observed during routine external examination undertaken as a part of the management of other research projects, one in 2006 and the other in 2011. Apart from the skin lesions, both birds showed no signs of illness and the lesions resolved spontaneously over a 2-month period. PATHOLOGICAL FINDINGS: The first case showed several 3-mm diameter firm, brown nodules located on the skin below the hock of both legs. The second case had a single multinodular mass that measured 7×20 mm, on the base of the bill. A portion of the mass and scab samples were collected for diagnosis. Histological examination of the nodules revealed severe ballooning degeneration of keratinocytes and epithelial hyperplasia. Round eosinophilic structures resembling avipoxvirus (APV) intracytoplasmic inclusion bodies (Bollinger bodies) were observed in the layers of keratinocytes. In deeper layers of the epidermis, there was evidence of secondary bacterial growth and inflammation. DIAGNOSIS: DNA was extracted from tissue samples and subjected to PCR analysis. Avipoxvirus 4b core protein gene was detected in both samples by PCR. Bootstrap analysis of APV 4b core protein gene revealed that APV isolates from two kiwi comprised two different subclades. One isolate displayed 100% sequence homology to subclade B1, and the other presented 100% sequence homology to subclade A3. CLINICAL RELEVANCE: This study confirmed that kiwi are susceptible to APV infection and that at least two different strains of APV are present in the population examined. Since there is no information on the origin, virulence, or prevalence of APV in kiwi, a seroprevalence study would be useful to elucidate the degree of exposure and immune response to the disease. This would allow a more informed approach to risk management of the disease in wild and captive populations.

Virus-like particles of hepatitis B virus core protein containing five mimotopes of infectious bursal disease virus (IBDV) protect chickens against IBDV

Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 μg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV.

Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes

Interferon-gamma (IFN-γ) has been reported to have antiviral activity against Hepatitis B virus (HBV) and to suppress HBV replication noncytolytically in vivo. Since systemic administration of IFN-γ may cause severe adverse effects, studies of the effects of liver-specific IFN-γ expression from adenoviral vectors in vivo have been investigated. In this study, a novel strategy has been described that drives specific expression of human IFN-γ in HBsAg-secreting hepatocytes. A bicistronic expression vector has been developed, pcDNA3.1-HBV antisense S gene-HCV core protein gene-HCV internal ribosome entry sites (IRES)-IFN-γ (pcDNA-SCIγ), by inserting four DNA fragments into pcDNA3.1. Tight modulation of HCV IRES-dependent translation by the HCV core protein was achieved using an antisense RNA technique with a bicistronic expression vector. HepG2 cells and HepG2.2.15 cells stably expressing HBV were transduced with pcDNA-SCIγ to test the responsiveness of IFN-γ to HBsAg expression. Gene transfer resulted in a low background and a 30-fold induction of IFN-γ expression from pcDNA-SCIγ in a cell-specific fashion. Hepatocyte-specific IFN-γ expression controlled effectively HBV replication in HBsAg-secreting HepG2.2.15 cells without cell toxicity.

Gliosis-specific transcription factor OASIS coincides with proteoglycan core protein genes in the glial scar and inhibits neurite outgrowth

OASIS gene, a member of the CREB/ATF transcription factor family, is upregulated in gliosis after CNS injury. However it remains to be determined how OASIS is implicated in gliotic reaction. In a glial scar, chondroitin sulfate proteoglycans (CSPGs) are also upregulated, which engenders the inhibition of axonal regeneration. We investigated the functional role of OASIS in gliosis in relation to CSPG core proteins that render lesions non-permissive for regenerating axons. We first examined the gene expression localization of OASIS using several markers in a cryo-injured mouse brain and compared the expression pattern of CSPG core protein genes with that of OASIS in a glial scar by double-labeling in situ hybridization. Our findings suggest that OASIS is induced in proximal reactive astrocytes that exhibit upregulated expression for CSPGs, including NG2 proteoglycan, versican, brevican, neurocan, and phosphacan core. Furthermore, the membrane fraction derived from OASIS-transfected C6 cells inhibits neurite outgrowth of NG108-15 cells, whereas its neurite outgrowth inhibitory effect is abrogated after chondroitinase ABC treatment. OASIS is likely to be involved in the regulatory mechanism of non-permissive environments for axonal outgrowth.

Phylogenetic analysis of VP1, VP2, and VP3 gene segments of genotype G5 group A rotavirus strains circulating in Brazil between 1986 and 2005

Genotype G5 group A rotavirus (RV-A), which is common in pigs and also detected in horses and cattle, circulated as endemic genotype in the 1980s and early 1990s in Brazil. After 1996, G5 RV-A has been replaced by G9 RV-A, becoming only sporadically detected. Recently, G5 has been reported in children with severe diarrhea in Argentina, Cameroon, Paraguay, People's Republic of China, and Vietnam, suggesting that, although uncommon in humans, it has a worldwide distribution. In a previous study, Brazilian G5 RV-A VP7 gene analysis demonstrated the existence of three main lineages: I, II, and III; all Brazilian strains and three porcine strains from Thailand grouped inside Lineage I. The VP8* subunit of VP4 gene showed that all P[8] strains fell into three major genetic lineages: P[8]-1; P[8]-2; and P[8]-3. Partial sequencing and phylogenetic analysis of VP1, VP2 and VP3 genes of P[8]G5 human RV-A strains were determined from 28 Brazilian strains collected from 1986 to 2005. The VP1-VP3 partial sequences analysis showed that the Brazilian strains have high amino acid identity with the human RV-A prototype IAL28 and other Wa-like genogroup strains. It was also shown that G5 RV-A Brazilian VP1–VP3 and VP7 sequences have a similar pattern of gouping: The study strains and the G5 prototype strain IAL-28 grouped together, while other prototypes, like OSU grouped separately. These results suggest that the core protein genes (VP1–VP3) of the G5 RV-A Brazilian strains might have originated from porcine and human strains. Phylogenetic analyses of VP7, VP4, VP1, VP2, and VP3 genes of P[8]G5 strains revealed a conserved genomic constellation (G5-P[8]-R1-C1-M1) with sequence similarity to Wa-like strains: IAL28, Wa, BE00048, CK00032, CK00033, DC4772 and DC1898, suggesting that despite the differences in genotypes (i.e., G5, G1 and G3) these viruses are genetically similar. The results presented here are fundamental to understand the epidemiology and evolution of genotype G5 RV-A and demonstrate the importance of continuous monitoring and molecular characterization of RV-A strains circulating in human and animal populations.

Stem cell genes are poorly expressed in chondrocytes from microtic cartilage

This study was aimed to see the difference between chondrocytes from normal cartilage compared to chondrocytes from microtic cartilage. Specific attentions were to characterize the growth of chondrocytes in terms of cell morphology, growth profile and RT-PCR analysis. Laboratory experiment using auricular chondrocytes. Chondrocytes were isolated from normal and microtic human auricular cartilage after ear reconstructive surgeries carried out at the Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured in vitro and subcultured until passage 4. Upon confluency, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and subjected to growth profile and gene expression analyses. Comparison was made between the microtic and normal chondrocytes. For growth profile analysis cell viability did not show significant differences between both samples. There are no significance differences between both samples in terms of its growth rate, except in passage 1 where microtic chondrocytes were significant lower in their growth rate. Population doubling time and total number of cell doubling of all samples also did not show any significant differences. Gene expression is measured using Real Time-Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). There is no significant differences in the expression of collagen type I, collagen type II, collagen type X, aggrecan core protein, elastin and sox9 genes in both samples. There are significant lower in the expression of sox2, nestin, BST-1 and OCT-4 gene in microtic chondrocytes compared to the normal chondrocytes. Stem cells markers are included in this study as stemness in cells may imply a greater proliferative potential and plasticity in vitro. Chondrocytes from microtic samples have the same properties as chondrocytes from normal samples and hold promises to be used as a starting material in the reconstruction of the external ear in future clinical application. The reduction in sox2, nestin, BST-1 and OCT-4 gene expression in microtic samples could be the possible cause of the arrested development of the external ear.

Avian Pox Infection in a Free-Living Crested Serpent Eagle (Spilornis cheela) in Southern Taiwan

Avian pox viruses (APVs) have been reported to cause infection in diverse avian species worldwide. Herein we report the first case of APV infection in a free-living bird, a subadult crested serpent eagle (Spilornis cheela), in Taiwan. In addition to the typical wart-like lesions distributed on the cere, eyelid, and face, there were also yellowish nodules below the tongue and on the hard palate. Phylogenetic analysis of the 4b core protein gene showed that the APV is very close to that found in white-tailed sea eagle (Haliaeetus albicilla) in Japan recently. Because both cases are located on the same major flyway for migratory birds, the impact of this virus with regard to the wild and migratory raptor species along the East Asian-Australasian Flyway and West Pacific Flyway requires immediate investigation.

The phylogenetic analysis of avipoxvirus in New Zealand

Avipoxvirus is known to be endemic in New Zealand and it is a cause of ongoing mortalities in the endangered black robin and shore plover populations. There is no information on the strains of avipoxvirus occurring in New Zealand and their likely origin or pathogenicity. This study was designed to identify the phylogenetic relationships of pathogenic avipoxvirus strains infecting introduced, native, and endemic bird species in New Zealand. Avipoxvirus 4b core protein gene was detected in tissue samples from 25/48 birds (52.1%) from 15 different species in New Zealand. Bootstrap analysis of avipoxvirus 4b core protein gene revealed that the New Zealand avipoxvirus isolates comprised of three different subclades. The majority of New Zealand avipoxvirus isolates (74%) belonged to A1 subclade which shared 100% genetic similarity with the fowlpox HPB strain. An isolate from a wood-pigeon (kereru) belonged to subclade A3, displaying 100% sequence homology to albatrosspox virus. An additional group, isolated from two shore plovers and one South Island saddleback, grouped within subclade B1 and presented 99% sequence homology to European PM33/2007 and Hawaiian HAAM 22.10H8 isolates. The results suggest that a variety of New Zealand bird species are susceptible to avipoxvirus infection, that there are more than two distinctive avipoxvirus subclades in New Zealand, and that the most prevalent A1 strain may have been introduced to New Zealand through introduced avian hosts such as passerines or poultry.

Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds.Methodology/Principal FindingsHere, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study.Conclusions/SignificanceWe anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

Avipoxvirus in great tits (Parus major)

A total of six cases and 37 suspect cases of skin avipoxvirus infection in great tits (Parus major) have been described in central Europe since 2005. Most of the cases were diagnosed during the winter season, from October to March. Analyses of the 4b core protein gene showed identical or almost identical DNA sequence in six isolates (one from Austria, three from Hungary, and two from Czech Republic). A morphogenesis of the avipoxvirus including a constitution of acidophilic-type inclusions (ATIs) was documented by electron microscopy in cells from lesions on great tits found in Czech Republic. Moreover, the ATI body protein gene was demonstrated using polymerase chain reaction in the isolate that caused ATIs. A number of new cases of poxvirus infection in great tits have emerged in central Europe since 2005, and the reason for this sudden increase remains unknown.

Poxvirus Infection in an American Red Squirrel (Tamiasciurus hudsonicus) from Northwestern Canada

There are two recognized poxviruses that are associated with disease in tree squirrels: squirrel fibroma virus (SQFV), Leporipoxvirus, which affects eastern grey squirrels (Sciurus carolinensis) in eastern North America, and squirrelpox virus (SQPV), a member of a newly identified poxvirus genus, which affects European red squirrels (Sciurus vulgaris) in the United Kingdom. In August 2008, a cutaneous poxvirus-associated disease was identified in a North American red squirrel (Tamiasciurus hudsonicus) from the Yukon Territory, Canada. The gross and microscopic appearance of the skin lesions was more consistent with SQPV than SQFV, and electron microscopy revealed poxvirions only within epithelial cells. Polymerase chain reaction (PCR) was used to identify poxvirus core protein encoding DNA in skin samples, and phylogenetic analysis showed that the inferred amino acid sequence was distinct from all other poxvirus species for which the core protein gene has been sequenced, including those of the genus Leporipoxvirus. Although the core protein sequence of SQPV was not available, comparison of the constructed phylogenetic tree to other published trees, based on major outer envelope proteins, revealed that the identified sequence occupies a position similar to SQPV in terms of its relationship to other poxviruses. However, PCR primers designed to amplify gene sequences encoding the SQPV major envelope protein and RNA polymerase did not amplify any sequences from infected tissues. These findings suggest that the virus present in this squirrel is a novel poxvirus of North American red squirrels. To our knowledge, this is the first case of poxvirus infection in Canadian squirrels outside of Ontario.

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

Towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development

Manipulation of the coronavirus genome to accommodate and express foreign genes is an attractive approach for gene delivery and vaccine development. By using an infectious cloning system developed recently for the avian coronavirus infectious bronchitis virus (IBV), the enhanced green fluorescent protein (EGFP) gene, the firefly luciferase gene and several host and viral genes (eIF3f, SARS ORF6, Dengue virus 1 core protein gene) were inserted into various positions of the IBV genome, and the effects on gene expression, virus recovery, and stability in cell culture were studied. Selected viruses were also inoculated into chicken embryos for studies of foreign gene expression at different tissue level. The results demonstrated the stability of recombinant viruses depends on the intrinsic properties of the foreign gene itself as well as the position at which the foreign genes were inserted. For unstable viruses, the loss of expression of the inserted genes was found to result from a large deletion of the inserted gene and even IBV backbone sequences. This represents a promising system for development of coronavirus-based gene delivery vectors and vaccines against coronavirus and other viral infections in chicken.

Poxvirus infection in Hungarian great tits ( Parus major ): Case report

From a total of 1819 great tits (Parus major) ringed in 2007 in Pilis Mountains, Hungary, 15 birds presented nodular proliferative lesions on different areas of the head and eyelids, suggesting a poxvirus infection. Three birds were submitted for analysis. The presence of avipoxvirus infection was confirmed by histopathology, electron microscopy (EM) and a polymerase chain reaction (PCR) based technique. Nucleotide sequence analysis of a 428 base pairs (bp) fragment of the viral 4b core protein gene revealed 100% identity between two of the Hungarian isolates (PM9 HUN, PM33 HUN) and two great tit poxvirus strains isolated in Norway in 1973 (GTV A256, GTV A311). The third Hungarian isolate (PM34 HUN) was more closely related to a different Norwegian isolate (GTVA310) than to the Hungarian isolates. The nucleotide sequence analysis of a shorter fragment of the viral 4b core protein (227 bp) gene revealed 100% identity between the Hungarian isolates, the same Norwegian isolates and a great tit poxvirus strain detected in Austria in 2007.

Hepatitis C virus ARFP/F protein interacts with cellular MM-1 protein and enhances the gene trans-activation activity of c-Myc

The ARFP/F protein is synthesized from the +1 reading frame of the hepatitis C virus (HCV) core protein gene. The function of this protein remains unknown. To study the function of the HCV ARFP/F protein, we have conducted the yeast two-hybrid screening experiment to identify cellular proteins that may interact with the ARFP/F protein. MM-1, a c-Myc interacting protein, was found to interact with HCV ARFP/F protein in this experiment. The physical interaction between ARFP/F and MM-1 proteins was further confirmed by the GST pull-down assay, the co-immunoprecipitation assay and confocal microscopy. As MM-1 can inhibit the gene transactivation activity of c-Myc, we have conducted further analysis to examine the possible effect of the ARFP/F protein on c-Myc. Our results indicate that the HCV ARFP/F protein can enhance the gene trans-activation activity of c-Myc, apparently by antagonizing the inhibitory effect of MM-1. The ability of the ARFP/F protein to enhance the activity of c-Myc raises the possibility that ARFP/F protein might play a role in hepatocellular transformation in HCV patients.

PPARα activation is essential for HCV core protein–induced hepatic steatosis and hepatocellular carcinoma in mice

Transgenic mice expressing HCV core protein develop hepatic steatosis and hepatocellular carcinoma (HCC), but the mechanism underlying this process remains unclear. Because PPARalpha is a central regulator of triglyceride homeostasis and mediates hepatocarcinogenesis in rodents, we determined whether PPARalpha contributes to HCV core protein-induced diseases. We generated PPARalpha-homozygous, -heterozygous, and -null mice with liver-specific transgenic expression of the core protein gene (Ppara(+/+):HCVcpTg, Ppara(+/-):HCVcpTg, and Ppara(-/-):HCVcpTg mice. Severe steatosis was unexpectedly observed only in Ppara(+/+):HCVcpTg mice, which resulted from enhanced fatty acid uptake and decreased mitochondrial beta-oxidation due to breakdown of mitochondrial outer membranes. Interestingly, HCC developed in approximately 35% of 24-month-old Ppara(+/+):HCVcpTg mice, but tumors were not observed in the other genotypes. These phenomena were found to be closely associated with sustained PPARalpha activation. In Ppara(+/-):HCVcpTg mice, PPARalpha activation and the related changes did not occur despite the presence of a functional Ppara allele. However, long-term treatment of these mice with clofibrate, a PPARalpha activator, induced HCC with mitochondrial abnormalities and hepatic steatosis. Thus, our results indicate that persistent activation of PPARalpha is essential for the pathogenesis of hepatic steatosis and HCC induced by HCV infection.

Diversity, origins and virulence of Avipoxviruses in Hawaiian Forest Birds

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.