Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

Latest Pleistocene—Holocene paleoceanographic trends on the continental margin of eastern Canada: Foraminiferal,dinoflagellate and pollen evidence

Micropaleontological studies were made of cores from four shelf basins on the eastern Canadian Margin: Emerald and Canso basins on the Scotian Shelf (44°–46° N), Notre Dame Channel, Newfoundland Shelf (50° N) and Cartwright Saddle, Labrador Shelf (55°). Events were correlated using a combination of¹⁴C dates and pollen stratigraphies. Surface- and bottom-water changes were compared on the basis of dinoflagellates and benthic foraminifera, respectively. The results indicate significant paleoceanographic shifts along a north—south gradient both prior to and during the Holocene.Distinct Late Pleistocene—Holocene paleoceanographic events were distinguished in the Emerald, Canso and Notre Dame basins; these events are less obvious in Cartwright Saddle which is in deeper water and further off-shore. Pleistocene glaciomarine sediments in all basins contain a fauna dominated byElphidium excavatum f.clavata; dinoflagellates and pollen are rare or absent. The widespreadElphidium fauna probably reflects turbid glacial meltwater and/or a permanent ice shelf cover from 20,000-10,000 yrs BP. The Notre Dame core also penetrates older sediment with an outer Labrador Current fauna which may represent a late Wisconsinian interstade at about 23,000 yrs BP. From 7,000–10,000 yrs BP a cold water fauna occurred which is similar to modern outer Labrador Current faunas. From about 5000–7000 yrs BP, a warm interval is indicated by a relatively warm-water calcareous benthonic foraminiferal fauna and increased representation of typical Gulf Stream dinoflagellates. The most recent change occurred in the last 2000 years with an abrupt cooling associated with stronger flow of the arctic inner Labrador Current. This cooling event is marked by an increase in arctic dinoflagellates and by an exclusively agglutinated benthonic foraminiferal fauna at two sites (Canso and Notre Dame). These Holocene paleoceanographic changes are not clearly seen in the benthic fauna of the deep northern basin (Cartwright Saddle) although dinoflagellate data at this site indicate that surface-water changes have occurred that are similar to those found in shallower basins.Shifts in the zonal position of the Gulf Stream and changes in the relative mass transports of the West Greenland and Labrador currents are mechanisms which may account for the paleoceanographic events. The glacial—interstadial—glacial sequence recorded in the Notre Dame Channel, in conjunction with other theories on glacial triggering mechanisms, provides biostratigraphic evidence which suggests the onset of a glacial stage in the near future.     

Arctic fox versus red fox in the warming Arctic: four decades of den surveys in north Yukon

During the last century, the red fox (Vulpes vulpes) has expanded its distribution into the Arctic, where it competes with the arctic fox (Vulpes lagopus), an ecologically similar tundra predator. The red fox expansion correlates with climate warming, and the ultimate determinant of the outcome of the competition between the two species is hypothesized to be climate. We conducted aerial and ground fox den surveys in the northern Yukon (Herschel Island and the coastal mainland) to investigate the relative abundance of red and arctic foxes over the last four decades. This region has undergone the most intense warming observed in North America, and we hypothesized that this climate change led to increasing dominance of red fox over arctic fox. Results of recent surveys fall within the range of previous ones, indicating little change in the relative abundance of the two species. North Yukon fox dens are mostly occupied by arctic fox, with active red fox dens occurring sympatrically. While vegetation changes have been reported, there is no indication that secondary productivity and food abundance for foxes have increased. Our study shows that in the western Arctic of North America, where climate warming was intense, the competitive balance between red and arctic foxes changed little in 40?years. Our results challenge the hypotheses linking climate to red fox expansion, and we discuss how climate warming’s negative effects on predators may be overriding positive effects of milder temperatures and longer growing seasons.     

Genetic Structure of Capelin (Mallotus villosus) in the Northwest Atlantic Ocean

Capelin (Mallotus villosus) is a commercially exploited, key forage-fish species found in the boreal waters of the North Pacific and North Atlantic Oceans. We examined the population structure of capelin throughout their range in the Canadian northwest Atlantic Ocean using genetic-based methods. Capelin collected at ten beach and five demersal spawning locations over the period 2002 through 2008 (N = 3,433 fish) were genotyped using six polymorphic microsatellite loci. Temporally distinct samples were identified at three beach spawning locations: Chance Cove, Little Lawn and Straitsview, Newfoundland. Four capelin stocks are assumed for fisheries management in the northwest Atlantic Ocean based on meristics, morphometrics, tag returns, and seasonal distribution patterns. Our results suggested groupings that were somewhat different than the assumed structure, and indicate at least seven genetically defined populations arising from two ancestral populations. The spatial mosaic of capelin from each of the two basal cluster groups explains much of the observed geographic variability amongst neighbouring samples. The genetic-defined populations were resolved at Jost’s D _est ≥ 0.01 and were composed of fish collected 1) in the Gulf of St. Lawrence, 2) along the south and east coasts of Newfoundland, 3) along coastal northern Newfoundland and southern Labrador, 4) along coastal northern Labrador, 5) near the Saguenay River, and at two nearshore demersal spawning sites, 6) one at Grebes Nest off Bellevue Beach on the east coast of Newfoundland, and 7) one off the coast of Labrador at Domino Run. Moreover, the offshore demersal spawners on the Scotian Shelf and Southeast Shoal appeared to be related to the inshore demersal spawners at Grebes Nest and in Domino Run and to beach spawners from the Gulf of St. Lawrence.     

Rabies emergence among foxes in Turkey

Sixteen rabies isolates recently collected from mainland Turkey and two isolates held within a British archive were used to form a representative cohort from a range of vectors, and were analyzed to identify potential causes for an increase of rabies within the fox (Vulpes vulpes) population in Turkey. Each isolate was characterized by sequence analysis of the nucleoprotein gene and compared phylogenetically to the cohort, to isolates from neighboring countries and to isolates from continental Europe and Russia. From this analysis the isolates could be divided into three groups associated with geographic location. This included a western group, an eastern group, and one isolate that did not group with any other Turkish isolate. This observation was also found using the heteroduplex mobility assay as an alternative method for typing rabies virus isolates. Further comparison with isolates from neighboring countries suggests that this isolate was related to viruses present in Georgia and could represent a recent import to Turkey from that country. Within the two larger groups, sequence data were obtained from both infected dogs and foxes suggesting that there has been transmission of virus between these two species. The direction of transmission could not be identified by the phylogenetic analysis, although absence of rabies within the fox population in previous years suggests that this could represent a recent spillover from the domestic dog to the fox.     

Concluding Remarks

Rabies seems to persist throughout most arctic regions, and the northern parts of Norway, Sweden and Finland, is the only part of the Arctic where rabies has not been diagnosed in recent time. The arctic fox is the main host, and the same arctic virus variant seems to infect the arctic fox throughout the range of this species. The epidemiology of rabies seems to have certain common characteristics in arctic regions, but main questions such as the maintenance and spread of the disease remains largely unknown. The virus has spread and initiated new epidemics also in other species such as the red fox and the racoon dog. Large land areas and cold climate complicate the control of the disease, but experimental oral vaccination of arctic foxes has been successful. This article summarises the current knowledge and the typical characteristics of arctic rabies including its distribution and epidemiology.     

Immunization of arctic foxes (Alopex lagopus) with oral rabies vaccine

Arctic foxes (Alopex lagopus) were successfully immunized against rabies using an orally-administered, liquid SAD-BHK21 live virus vaccine in a sausage bait. Immunization was determined by serologic response and by resistance to challenge with an arctic rabies virus strain. Virus was not shed in saliva following oral vaccination, indicating that arctic foxes would not infect other foxes after ingesting this vaccine. High antibody levels were present in all experimental foxes 2 wk following initial vaccination. A booster vaccination at 56 wk induced a significant serologic response within 1 wk, suggesting an anamnestic response but titers began to decline within 8 wk in most foxes. Foxes were observed for 16 mo following the challenge and exhibited no symptoms of rabies. The SAD-BHK21 rabies vaccine in a sausage bait system has a strong potential for vaccinating wild populations of arctic fox.     

Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska

For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine‐scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.     

Rough-Legged Buzzards,Arctic Foxes and Red Foxes in a Tundra Ecosystem without Rodents

Small rodents with multi-annual population cycles strongly influence the dynamics of food webs, and in particular predator-prey interactions, across most of the tundra biome. Rodents are however absent from some arctic islands, and studies on performance of arctic predators under such circumstances may be very instructive since rodent cycles have been predicted to collapse in a warming Arctic. Here we document for the first time how three normally rodent-dependent predator species—rough-legged buzzard, arctic fox and red fox – perform in a low-arctic ecosystem with no rodents. During six years (in 2006-2008 and 2011-2013) we studied diet and breeding performance of these predators in the rodent-free Kolguev Island in Arctic Russia. The rough-legged buzzards, previously known to be a small rodent specialist, have only during the last two decades become established on Kolguev Island. The buzzards successfully breed on the island at stable low density, but with high productivity based on goslings and willow ptarmigan as their main prey – altogether representing a novel ecological situation for this species. Breeding density of arctic fox varied from year to year, but with stable productivity based on mainly geese as prey. The density dynamic of the arctic fox appeared to be correlated with the date of spring arrival of the geese. Red foxes breed regularly on the island but in very low numbers that appear to have been unchanged over a long period – a situation that resemble what has been recently documented from Arctic America. Our study suggests that the three predators found breeding on Kolguev Island possess capacities for shifting to changing circumstances in low-arctic ecosystem as long as other small - medium sized terrestrial herbivores are present in good numbers.     

Molecular epidemiology of terrestrial rabies in the former Soviet Union

Fifty-five rabies virus isolates originating from different regions of the former Soviet Union (FSU) were compared with isolates originating from Eurasia, Africa, and North America according to complete or partial nucleoprotein (N) gene sequences. The FSU isolates formed five distinct groups. Group A represented viruses originating from the Arctic, which were similar to viruses from Alaska and Canada. Group B consisted of "Arctic-like" viruses, originating from the south of East Siberia and the Far East. Group C consisted of viruses circulating in the steppe and forest-steppe territories from the European part of Russia to Tuva and in Kazakhstan. These three phylogenetic groups were clearly different from the European cluster. Viruses of group D circulate near the western border of Russia. Their phylogenetic position is intermediate between group C and the European cluster. Group E consisted of viruses originating from the northwestern part of Russia and comprised a "northeastern Europe" group described earlier from the Baltic region. According to surveillance data, a specific host can be defined clearly only for group A (arctic fox; Alopex lagopus) and for the Far Eastern part of the group B distribution area (raccoon dog; Nyctereutes procyonoides). For other territories and rabies virus variants, the red fox (Vulpes vulpes) is the main virus reservoir. However, the steppe fox (Vulpes corsac), wolf (Canis lupus), and raccoon dog are also involved in virus circulation, depending on host population density. These molecular data, joined with surveillance information, demonstrate that the current fox rabies epizootic in the territory of the FSU developed independently of central and western Europe. No evidence of positive selection was found in the N genes of the isolates. In the glycoprotein gene, evidence of positive selection was strongly suggested in codons 156, 160, and 183. At these sites, no link between amino acid substitutions and phylogenetic placement or specific host species was detected.     

Natal den selection by sympatric arctic and red foxes on Herschel Island,Yukon, Canada

In the twentieth century, red fox (Vulpes vulpes) expanded into the Canadian Arctic, where it competes with arctic fox (Vulpes lagopus) for food and shelter. Red fox dominates in physical interactions with the smaller arctic fox, but little is known about competition between them on the tundra. On Hershel Island, north Yukon, where these foxes are sympatric, we focused on natal den choice, a critical aspect of habitat selection. We tested the hypothesis that red fox displaces arctic fox from dens in prey-rich habitats. We applied an approach based on model comparisons to analyse a 10-year data set and identify factors important to den selection. Red fox selected dens in habitats that were more prey-rich in spring. When red foxes reproduced, arctic fox selected dens with good springtime access, notably many burrows unblocked by ice and snow. These provided the best refuge early in the reproductive season. In the absence of red foxes, arctic foxes selected dens offering good shelter (i.e. large isolated dens). Proximity to prey-rich habitats was consistently less important than the physical aspects of dens for arctic fox. Our study shows for the first time that red foxes in the tundra select dens associated primarily with prey-rich areas, while sympatric arctic foxes do not. These results fit a model of red fox competitively interfering with arctic fox, the first detailed study of such competition in a true arctic setting.     

Exclusion by interference competition? The relationship between red and arctic foxes

The distribution of many predators may be limited by interactions with larger predator species. The arctic fox in mainland Europe is endangered, while the red fox is increasing its range in the north. It has been suggested that the southern distribution limit of the arctic fox is determined by interspecific competition with the red fox. This has been criticised, on the basis that the species co-exist on a regional scale. However, if the larger red fox is superior and interspecific competition important, the arctic fox should avoid close contact, especially during the breeding season. Consequently, the distribution of breeding dens for the two species would be segregated on a much smaller spatial and temporal scale, in areas where they are sympatric. We tested this hypothesis by analysing den use of reproducing arctic and red foxes over 9 years in Sweden. High quality dens were inhabited by reproducing arctic foxes more often when no red foxes bred in the vicinity. Furthermore, in two out of three cases when arctic foxes did reproduce near red foxes, juveniles were killed by red foxes. We also found that breeding arctic foxes occupied dens at higher altitudes than red foxes did. In a large-scale field experiment, red foxes were removed, but the results were not conclusive. However, we conclude that on the scale of individual territories, arctic foxes avoid areas with red foxes. Through interspecific interference competition, the red fox might thus be excluding the arctic fox from breeding in low altitude habitat, which is most important in years when food abundance is limited and competition is most fierce. With high altitude refuges being less suitable, even small-scale behavioural effects could scale up to significant effects at the population level.     

Elimination of rabies from red foxes in eastern Ontario

The province of Ontario (Canada) reported more laboratory confirmed rabid animals than any other state or province in Canada or the USA from 1958-91, with the exception of 1960-62. More than 95% of those cases occurred in the southern 10% of Ontario (approximately 100,000 km2), the region with the highest human population density and greatest agricultural activity. Rabies posed an expensive threat to human health and significant costs to the agricultural economy. The rabies variant originated in arctic foxes: the main vector in southern Ontario was the red fox (Vulpes vulpes), with lesser involvement of the striped skunk (Mephitis mephitis). The Ontario Ministry of Natural Resources began a 5 yr experiment in 1989 to eliminate terrestrial rabies from a approximately 30,000 km2 study area in the eastern end of southern Ontario. Baits containing oral rabies vaccine were dropped annually in the study area at a density of 20 baits/km2 from 1989-95. That continued 2 yr beyond the original 5 yr plan. The experiment was successful in eliminating the arctic fox variant of rabies from the whole area. In the 1980's, an average of 235 rabid foxes per year were reported in the study area. None have been reported since 1993. Cases of fox rabies in other species also disappeared. In 1995, the last bovine and companion animal cases were reported and in 1996 the last rabid skunk occurred. Only bat variants of rabies were present until 1999, when the raccoon variant entered from New York (USA). The success of this experiment led to an expansion of the program to all of southern Ontario in 1994. Persistence of terrestrial rabies, and ease of elimination, appeared to vary geographically, and probably over time. Ecological factors which enhance or reduce the long term survival of rabies in wild foxes are poorly understood.     

Rabies and canine distemper in an arctic fox population in Alaska

Two oil field workers were attacked by a rabid arctic fox (Alopex lagopus) in the Prudhoe Bay oil field (Alaska, USA) prompting officials to reduce the local fox population. Ninety-nine foxes were killed during winter 1994. We tested foxes for prevalence of rabies and canine distemper. Exposure to rabies was detected in five of 99 foxes. Of the five, only one fox had rabies virus in neural tissue as determined by the direct fluorescent antibody test. The other four foxes had been exposed to rabies, but had apparently produced antibodies and did not have an active infection. No evidence of canine distemper was detected as determined by the absence of distemper antibodies in serum and distemper virus in neural tissue.     

Prevalence of Trichinella spiralis in black bears (Ursus americanus) from Newfoundland and Labrador, Canada.

Tongue and diaphragm samples from 158 black bears (Ursus americanus) from Newfoundland and Labrador were examined for Trichinella spiralis. No larvae were detected in samples from the island of Newfoundland but one animal from the Labrador samples was infected. The results of this and other studies suggest a lack of involvement of the black bear in a sylvatic cycle of T. spiralis in eastern Canada.     

Serologic evidence of arbovirus infections in humans and wild animals in Alaska

Blood samples were collected from humans and several species of free-ranging wild animals in Alaska. Sera were tested for antibody to Jamestown Canyon (JC), snowshoe hare (SSH), Northway (NOR), Klamath (KLA), Sakhalin (SAK), Great Island (GI), and Silverwater (SIL) virus. JC antibody was found in 54% of 121 human, 89% of 97 bison (Bison bison), 51% of 84 Dall sheep (Ovis dalli), 43% of 68 snowshoe hare (Lepus americanus), and 3% of 33 arctic fox (Alopex lagopus) sera. SSH antibody was found in 42% of 121 human, 89% of 97 bison, 41% of 84 Dall sheep, and 65% of 68 snowshoe hare sera. NOR antibody was found in 14% of 121 human, 94% of 97 bison, 84% of 84 Dall sheep, 43% of 69 caribou (Rangifer tarandus), 3% of 68 snowshoe hare, 48% of 64 grizzly bear (Ursus arctos), 3% of 33 arctic fox, and 78% of 27 moose (Alces alces) sera. KLA antibody was found in 5% of 121 human and 40% of 97 bison sera. SAK antibody was found in 2% of 97 bison and 3% of 33 arctic fox sera. GI antibody was found in 1% of 97 bison sera. No SIL antibody was found in any sera tested. Thus the natural host ranges of JC, SSH, NOR, and KLA viruses have been extended by inference from the occurrence of antibody.     

Ecological–genetic approach in modeling the natural evolution of a population: Prospects and special aspects of verification

This study presents a complex approach for modeling the natural evolution of a population in terms of population number and dynamics of the genetic structure. A set of dynamic models that consider various types of natural selection was applied to describe possible mechanisms underlying the formation of existing genetic variations in litter sizes in coastal, inland, and farmed arctic fox populations (Alopex lagopus, family Canidae, order Carnivora). The r–K selection model for uniform population and the models with natural selection were assessed on various life cycle stages in a two-age population. The life cycle of arctic fox was fitted to the population model with two age stages. The different reproductive potentials and survivability of progeny on the early stage of life cycle were genetically determined using the model with a single diallelic gene. A monomorphism was obtained for a considered characteristic in a population of coastal arctic fox with constant food supply. Meanwhile, a polymorphism with cyclic fluctuations in population number and gene frequency was obtained in inland arctic fox populations, which could be due to cyclic fluctuations of prey. In farmed fox populations, the considered gene becomes pleiotropic (defines the survival rate of individuals on early and late stages of the life cycle) because of artificial selection performed by farmers to increase the reproductive success of breeders. The application of an appropriate model (with selection by pleiotropic gene) can be used to determine the elimination rate of low litter size alleles from the farmed populations. The possible applications of the proposed models for formulating and solving optimal control tasks in arctic fox populations are discussed too.     

Emergency rabies control in a community of two high-density hosts

ABSTRACT: BACKGROUND: Rabies is a fatal viral disease that potentially can affect all mammals. Terrestrial rabies is not present in the United Kingdom and has been eliminated from Western Europe. Nevertheless the possibility remains that rabies could be introduced to England, where it would find two potentially suitable hosts, red foxes and badgers. With the aim to analyse the spread and emergency control of rabies in this two species host community, a simulation model was constructed. Different control strategies involving anti-rabies vaccination and population culling were developed, considering control application rates, spatial extent and timing. These strategies were evaluated for efficacy and feasibility to control rabies in hypothetical rural areas in the South of England immediately after a disease outbreak. RESULTS: The model confirmed that both fox and badger populations, separately, were competent hosts for the spread of rabies. Realistic vaccination levels were not sufficient to control rabies in high-density badger populations. The combined species community was a very strong rabies host. However, disease spread within species appeared to be more important than cross-species infection. Thus, the drivers of epidemiology depend on the potential of separate host species to sustain the disease. To control a rabies outbreak in the two species, both species had to be targeted. Realistic and robust control strategies involved vaccination of foxes and badgers, but also required badger culling. Although fox and badger populations in the UK are exceptionally dense, an outbreak of rabies can be controlled with a higher than 90% chance, if control response is quick and follows a strict regime. This requires surveillance and forceful and repeated control campaigns. In contrast, an uncontrolled rabies outbreak in the South of England would quickly develop into a strong epizootic involving tens of thousands of rabid foxes and badgers. CONCLUSIONS: If populations of both host species are sufficiently large, epizootics are driven by within-species transmission, while cross-species-infection appears to be of minor importance. Thus, the disease control strategy has to target both host populations.     

Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission

One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.