Hawaii Strain Research Articles

Generation of Mild Recombinants of Papaya Ringspot Virus to Minimize the Problem of Strain-Specific Cross-Protection.

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain, PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross-protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes, of which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered to result from nitrous acid induction because 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3' fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3' regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3', with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3' retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3' fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.

Natural variation in the roles of C. elegans autophagy components during microsporidia infection.

Natural genetic variation can determine the outcome of an infection, and often reflects the co-evolutionary battle between hosts and pathogens. We previously found that a natural variant of the nematode Caenorhabditis elegans from Hawaii (HW) has increased resistance against natural microsporidian pathogens in the Nematocida genus, when compared to the standard laboratory strain of N2. In particular, HW animals can clear infection, while N2 animals cannot. In addition, HW animals have lower levels of initial colonization of Nematocida inside intestinal cells, compared to N2. Here we investigate how this natural variation in resistance relates to autophagy. We found that there is much better targeting of autophagy-related machinery to parasites under conditions where they are cleared. In particular, ubiquitin targeting to Nematocida cells correlates very well with their subsequent clearance in terms of timing, host strain and age, as well as species of Nematocida. Furthermore, clearance correlates with targeting of the LGG-2/LC3 autophagy protein to parasite cells, with HW animals having much more efficient targeting of LGG-2 to parasite cells than N2 animals. Surprisingly, however, we found that LGG-2 is not required to clear infection. Instead, we found that LGG-2/LC3 regulates Nematocida colonization inside intestinal cells. Interestingly, LGG-2/LC3 regulates intracellular colonization only in the HW strain, and not in N2. Altogether these results demonstrate that there is natural genetic variation in an LGG-2-dependent process that regulates microsporidia colonization inside intestinal cells, although not microsporidia clearance.

Genome characterization and distribution of Taro bacilliform CH virus on taro in Hawaii, USA

Taro bacilliform CH virus (TaBCHV) is the second badnavirus found infecting taro (Colocasia esculenta), after Taro bacilliform virus (TaBV). We determined the complete genome of TaBCHV Hawaii Strain (KY359389) from taro in Hawaii and examined its occurrence and distribution. Alignment analysis showed the TaBCHV Hawaii Strain has 93% and 94% sequence identity to China strains TaBCHV-1 and TaBCHV-2 respectively. Phylogenetic analysis basing on the whole genome sequence of 21 selected badnaviruses, showed that the TaBCHV Hawaii Strain clustered within the same branch as the two China strains. All sequences from available TaBCHV strains are most closely related to Piper yellow mottle virus (PYMoV; NC022365) and are most distant from TaBV (NC004450). We used RT-PCR to determine the occurrence and distribution of TaBCHV on taro in Hawaii. The positive rates of the virus on the five islands were 72% on Oahu, 53% on Hawaii, 89% on Maui, 73% on Molokai, and 78% on Kauai, with a mean positive rate of 73%. This is the first report of the complete genome sequence and the first report of the occurrence of this virus in the United States.

Molecular Characterization and Distribution of Two Strains of Dasheen mosaic virus on Taro in Hawaii.

Dasheen mosaic virus (DsMV) is one of the major viruses affecting taro (Colocasia esculenta) production worldwide. Whole genome sequences were determined for two DsMV strains, Hawaii Strain I (KY242358) and Hawaii Strain II (KY242359), from taro in Hawaii. They represent the first full-length coding sequences of DsMV reported from the United States. Hawaii Strains I and II were 77 and 85% identical, respectively, with other completely sequenced DsMV isolates. Hawaii Strain I was most closely related to vanilla mosaic virus (VanMV) (KX505964.1), a strain of DsMV infecting vanilla in the southern Pacific Islands. Hawaii Strain II was most closely related to a taro DsMV isolate CTCRI-II-14 (KT026108.1) from India. Phylogenetic analysis of all available DsMV isolates based on amino acid sequences of their coat protein showed some correlation between host plant and genetic diversity. Analyses of DsMV genome sequences detected three recombinants from China and India among the six isolates with known complete genome sequences. The DsMV strain NC003537.1 from China is a recombinant of KJ786965.1 from India and Hawaii Strain II. Another DsMV strain KT026108.1 is a recombinant of Hawaii Strain II and NC003537.1 from China. The third DsMV strain KJ786965.1 from India is a recombinant of Hawaii Strain II and NC003537.1 from China. To our knowledge, this is the first report of recombination events in DsMV. Both Hawaii Strains I and II of DsMV were found widespread throughout the Hawaiian islands.

Production Comparison of Three Genetic Strains of Freshwater Prawn, Macrobrachium rosenbergii, Raised Under Two Pond Management Technologies

AbstractThree genetic strains (Texas [cultured], Hawaii [cultured], and Myanmar [wild]) of freshwater prawn, Macrobrachium rosenbergii, were characterized and compared under two pond grow‐out management technologies using a 3 × 2 factorial design. Juvenile prawns (45 d nursed juveniles) from each strain were stocked at individual average weights of 0.4 ± 0.3 g (Texas), 0.3 ± 0.2 g (Hawaii), and 0.3 ± 0.2 g (Myanmar). The low input management technology prawns were stocked at 24,700 /ha with no added substrate. The high input management technology prawns were stocked at 74,100 /ha with the addition of artificial substrate. Each of the six treatment combinations were replicated in three, 0.04 ha earthen ponds (total of 18 ponds). Prawns were fed a sinking pellet (32% protein) once daily at a standardized rate. After 112 d, prawns were harvested, bulk weighed, and counted. Survival of Texas strain (95%) was significantly higher (P ≤ 0.05) than Myanmar strain (77–80%) under both management technologies with survival of Hawaii strain (86–91%) intermediate and not significantly different (P > 0.05) from other strains. Under both management technologies, average weight, total production, and marketable percentage (>20 g) was significantly better (P ≤ 0.05) in Texas and Hawaii strains in comparison to the Myanmar strain. These data appear to indicate that the cultured strains evaluated in this study demonstrate positive impacts of domestication and do not indicate inbreeding depression.

Multigenic Natural Variation UnderliesCaenorhabditis elegansOlfactory Preference for the Bacterial PathogenSerratia marcescens

The nematode Caenorhabditis elegans can use olfaction to discriminate among different kinds of bacteria, its major food source. We asked how natural genetic variation contributes to choice behavior, focusing on differences in olfactory preference behavior between two wild-type C. elegans strains. The laboratory strain N2 strongly prefers the odor of Serratia marcescens, a soil bacterium that is pathogenic to C. elegans, to the odor of Escherichia coli, a commonly used laboratory food source. The divergent Hawaiian strain CB4856 has a weaker attraction to Serratia than the N2 strain, and this behavioral difference has a complex genetic basis. At least three quantitative trait loci (QTLs) from the CB4856 Hawaii strain (HW) with large effect sizes lead to reduced Serratia preference when introgressed into an N2 genetic background. These loci interact and have epistatic interactions with at least two antagonistic QTLs from HW that increase Serratia preference. The complex genetic architecture of this C. elegans trait is reminiscent of the architecture of mammalian metabolic and behavioral traits.

Thermal Death Kinetics of Mediterranean, Malaysian, Melon, and Oriental Fruit Fly (Diptera: Tephritidae) Eggs and Third Instars

The late-aged egg and third-instar life stages of laboratory-reared Malaysian fruit fly, Bactrocera latifrons (Hendel); Mediterranean fruit fly, Ceratitis capitata (Wiedemann); melon fly, B. cucurbitae Coquillett; and oriental fruit fly, B. dorsalis (Hendel), (Diptera: Tephritidae); and the third instars of wild Mediterranean fruit fly were exposed to thermal treatments. A heating block system was used to determine the thermal death kinetics of the four fruit fly species. Treatments consisted of heating the fruit fly life stages to 44, 46, 48, and 50 degrees C and holding for different times ranging from 0 to 120 min depending on the thermal mortality response and time required to obtain 100% mortality for each species and life stage. The 0.5-order kinetic model had the best fit to the survival ratio for all the treatment temperatures and was used to predict lethal times. The thermal death time (TDT) curves showed a tolerance order of Mediterranean fruit fly eggs < or = third instars at 44, 46, and 50 degrees C, third instars < or = eggs at 48 degrees C, and wild third instars < the laboratory-reared third instars. Comparison between Mediterranean fruit fly third instar thermotolerance from Hawaii and Israel showed that Israel Mediterranean fruit fly was more thermotolerant. A comparison of minimum treatment times at a given temperature required to obtain 100% mortality of laboratory-reared Malaysian, Mediterranean (Hawaii and Israel strains), melon, Mexican, and oriental fruit fly eggs or third instars and wild Mediterranean fruit fly (Hawaii strain) eggs or third instars showed that oriental fruit fly was the most thermotolerant among the third instars, and the difference in heat tolerance between third instars and eggs was negligible at 50 degrees C.

Bioinformatics analysis and characteristics of envelop glycoprotein E epitopes of dengue virus

The major envelope glycoprotein E of dengue (DEN) virus plays a central role in the biology of flaviviruses. It is capable of inducing a protective immune response in vivo and responsible for the viral binding to the cellular receptor. The crystal structures of glycoprotein E ectodomains have already been determined. However, it is still un-clear where the well-defined B-cell epitopes for glycoprotein E which induce the neutralizing an-tibodies locates. Thus, in order to characterize the role of glycoprotein E in the pathogenesis of dengue virus infection, we first used network servers (http://bio.dfci. harvard.edu/Tools/ & http://www. imtech. res. in) to predict and analyze the well defined B-cell and T-cell epitopes of the glycoprotein of the DEN-1 HAWAII strain. Then based on the highly conserved envelop glyco-protein amino acids, the hydrophilicity, antigenic-ity, accessibility and flexibility of envelop glyco-protein E were further predicted by using Biotic softwares (DNASTAR) and network servers (http://bio. dfci.harvard.edu/Tools/), the secondary structure was putatively obtained. In our study, the sequence at 281-295 amino acid (aa) for den-gue virus type 1 HAWAII strain and the sequence at 345-359, 383-397 for dengue virus type 2 NGC strain were predicted as the more prevalent epi-topes by using multiple parameters and different analysis softwares, respectively. Two epitopes of DEN-2 and one of DEN-1 locate on the domain Ш and domainⅡ of the protein E, respectively. Sub-sequently, further studies will be carried out to examine the antigenicity and protection of the synthetic peptides with higher scores in the av-erage antigen index (AI) and better hydrophilic properties determined by our data.

Insecticidal effect of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) against Tribolium confusum du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) in stored wheat

The confused flour beetle, Tribolium confusum, and the Mediterranean flour moth, Ephestia kuehniella, are important pests of stored grain products. The insecticidal effect of three strains (UK 76 [=Nemasys], USA/SC, and Hawaii) of the entomopathogenic nematode Steinernema feltiae was determined in the laboratory, in wheat, against these pests. The nematodes were applied at three dose rates: 100, 300 and 900 nematodes/insect individual. The Hawaii strain was most virulent against T. confusum adults and larvae, with a significant dose effect in the case of the larvae. Larval mortality of this species reached 79% and 100% after 7 and 14 d of exposure to the nematodes, respectively, at the highest dose applied. On the other hand, adult mortality of T. confusum did not exceed 66%. In the case of E. kuehniella larvae, USA/SC performed best causing 52% and 69% mortality after 7 and 14 d exposure, respectively, at the highest dose tested. Since very few data are available on the effect of entomopathogenic nematodes against these pests, it is concluded that the Hawaii and USA/SC strains of S. feltiae should be further investigated as promising biological control agents for T. confusum and E. kuehniella.

In vitro and in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain) and dengue 1 virus (Hawaii strain)

Two galactomannans, one extracted from seeds of Mimosa scabrella, having a mannose to galactose ratio of 1.1, and another with a 1.4 ratio from seeds of Leucaena leucocephala, were sulfated. The products from M. scabrella (BRS) and L. leucocephala (LLS) had a degree of sulfation of 0.62 and 0.50, and an average molecular weight of 620×10 3 and 574×10 3 g mol −1, respectively. Their activities against yellow fever virus (YFV; BeH111 strain) and dengue 1 virus (DEN-1; Hawaii strain) were evaluated. This was carried out in young mice following intraperitoneal infection with YFV. At a dose of 49 mg kg −1, BRS and LLS gave protection against death in 87.7 and 96.5% of the mice, respectively. When challenged with 37.5 LD50 of YFV, mice previously inoculated with BRS+virus or LLS+virus, showed 93.3 and 100% resistance, respectively, with neutralization titers similar to mice injected with 25 LD50 of formaldehyde-inactivated YFV. In vitro experiments with YFV and DEN-1 in C6/36 cell culture assays in 24-well microplates showed that concentrations that produced a 100-fold decrease in virus titer of YFV were 586 and 385 mg l −1 for BRS and LLS, respectively. For DEN-1 they were 347 and 37 mg l −1, respectively. Sulfated galactomannans, thus demonstrate in vitro and in vivo activity against flaviviruses.

Human Calicivirus Infection in Infants and Young Children of the Metropolitan Washington, DC Area

Sequential serum samples collected from individuals at approximately 3, 8, and 13 months of age who were enrolled in a rotavirus vaccine study conducted in Washington, DC from October 1989 through January 1990, were analyzed for the presence of antibodies to the Norwalk, Hawaii, and Toronto strains of human calicivirus by an enzyme-linked immunosorbent assay. Antibodies were detected to the Hawaii, Norwalk, and Toronto viruses in 29 (85.3%), 24 (70.6%), and 27 (79.4%) of the serum samples, respectively, obtained from 34 children approximately one year following the final dose of rotavirus vaccine. Evidence was found for a high infection rate with these three viruses, with the highest frequency of serologic responses detected between the 8- and 13-month time points. During this period, 14 (42.4%), 13 (39.4%), and 16 (48.5%) of the 33 children tested showed evidence for a serologic response to Hawaii virus, Norwalk virus, and Toronto virus, respectively, as evidence by a 4-fold increase in antibody titer. Our data indicate that human calicivirus infections are common in young individuals living in die metropolitan Washington, DC area.

Human Calicivirus Infection in Infants and Young Children of the Metropolitan Washington, DC Area

Sequential serum samples collected from individuals at approximately 3, 8, and 13 months of age who were enrolled in a rotavirus vaccine study conducted in Washington, DC from October 1989 through January 1990, were analyzed for the presence of antibodies to the Norwalk, Hawaii, and Toronto strains of human calicivirus by an enzyme-linked immunosorbent assay. Antibodies were detected to the Hawaii, Norwalk, and Toronto viruses in 29 (85.3%), 24 (70.6%), and 27 (79.4%) of the serum samples, respectively, obtained from 34 children approximately one year following the final dose of rotavirus vaccine. Evidence was found for a high infection rate with these three viruses, with the highest frequency of serologic responses detected between the 8- and 13-month time points. During this period, 14 (42.4%), 13 (39.4%), and 16 (48.5%) of the 33 children tested showed evidence for a serologic response to Hawaii virus, Norwalk virus, and Toronto virus, respectively, as evidence by a 4-fold increase in antibody titer. O...

Broad-Spectrum Resistance to Different Geographic Strains ofPapaya ringspot virusin Coat Protein Gene Transgenic Papaya

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.

Microalgae with enormous potential

Hawaiian biotech company Aquasearch and the University of Hawaii have joined forces in drug development. They hope to create novel pharmaceuticals based on chemicals produced by oceanic microalgae. The microalgae, found mainly in the Pacific Ocean basin, are proving to be a vast and untapped source of useful bioactive compounds. Under the recent agreement, Aquasearch will create compound libraries from chemicals produced by microalgae in the University of Hawaii's strain collection, with the University receiving royalties for potential new drugs. Researchers have already isolated two anti-cancer candidates. Past efforts to harvest useful amounts of compounds from microalgae were thwarted because the microscopic algae were difficult to culture in large quantities. However, using massive, computer-controlled photobioreactors, Aquasearch grows microalgae on an enormous scale. AVhttp://www.aqse.com/astax-3.htm

Caracterización molecular de la secuencia parcial del gen de la glicoproteína NS1 del virus dengue 1 proveniente de Máncora, Perú

A 419bp-NS1 genetic region corresponding to Dengue virus 1 was characterised from an outbreak in Mancora Piura. The comparison of nucleotide sequences revealed that Peruvian isolates showed high correlation (93.32%) with a Hawaii strain. Amino acids comparisons revealed non-conservative changes into humoral response epitope domains. On the other hand, the hydropathy profile of NS1 was similar to other referential strains. The results suggest that more comparisons are needed regarding genetic identity and mutations into the epitope domain of Peruvian dengue 1 virus.

Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells

The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells. Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression. The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells. There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein. Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study.

Comparison of antigenic relationships between Hawaii and Mexican strains of Snow Mountain virus-like human caliciviruses using baculovirus-expressed viral capsid antigens 855

Comparison of antigenic relationships between Hawaii and Mexican strains of Snow Mountain virus-like human caliciviruses using baculovirus-expressed viral capsid antigens 855

Prevalence of antibodies to the Hawaii strain of human calicivirus as measured by a recombinant protein based immunoassay

The evaluation of an enzyme immunoassay using recombinant Hawaii virus-like particles (rHV-LPs) with a panel of sera which had been screened previously for antibodies to Norwalk virus (NV) and Mexico virus (MxV) is described. The assay was also applied to study the epidemiology of Hawaii virus. Adult volunteers challenged with the prototype (genogroup II, human calicivirus) HV developed significant IgG responses (16-32 fold rises) following challenge whereas adults challenged or naturally infected with NV (genogroup I) did not. Lesser antibody responses (4-8 fold rises) were demonstrated in volunteers challenged with Snow Mountain agent (SMA) and patients infected by SRSV 'UK3' and 'UK4' strains, indicating a degree of antigenic relatedness among viruses within genogroup II. Comparison of the seroprevalence of IgG antibodies to rHV, rMxV and rNV in 338 children in London showed that infections with genogroup II viruses are prevalent and occur earlier in life than NV. Many young children had antibodies to MxV but not HV indicating that genogroup II viruses have both conserved and antigenically distinct epitopes. A serological study on 566 Canadians aged between 9 and 79 years showed that the prevalence of antibodies to rHV rose with age from 65-100% and from 53-100% for NV. Measurement of antibody response in a heart transplant patient infected with an MxV-like virus showed significant responses to both rMxV and rHV. Continuous monitoring of the patient over two years showed that antibody levels declined rapidly to prechallenge levels after a year.

Prevalence of antibodies to the Hawaii strain of human calicivirus as measured by a recombinant protein based immunoassay

The evaluation of an enzyme immunoassay using recombinant Hawaii virus–like particles (rHVLPs) with a panel of sera which had been screened previously for antibodies to Norwalk virus (NV) and Mexico virus (MxV) is described. The assay was also applied to study the epidemiology of Hawaii virus. Adult volunteers challenged with the prototype (genogroup II, human calicivirus) HV developed significant IgG responses (16–32 fold rises) following challenge whereas adults challenged or naturally infected with NV (genogroup I) did not. Lesser antibody responses (4–8 fold rises) were demonstrated in volunteers challenged with Snow Mountain agent (SMA) and patients infected by SRSV ‘UK3’ and ‘UK4’ strains, indicating a degree of antigenic relatedness among viruses within genogroup II. Comparison of the seroprevalence of Ig G antibodies to rHV, rMxV and rNV in 338 children in London showed that infections with genogroup II viruses are prevalent and occur earlier in life than NV. Many young children had antibodies to MxV but not HV indicating that genogroup II viruses have both conserved and antigenically distinct epitopes. A serological study on 566 Canadians aged between 9 and 79 years showed that the prevalence of antibodies to rHV rose with age from 65–100% and from 53–100% for NV. Measurement of antibody response in a heart transplant patient infected with an MxV-like virus showed significant responses to both rMxV and rHV. Continuous monitoring of the patient over two years showed that antibody levels declined rapidly to prechallenge levels after a year. J. Med. Virol. 54:135–139, 1998. © 1998 Wiley-Liss,Inc.

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus.

The complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10,326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3344 amino acids with a 5' leader of 85 nucleotides and a 3' non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3' non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5-89.8% nucleotide identity of their genes and 91.2-97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0-16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas.