Molecular epidemiology of enteroviruses causing uveitis and multisystem hemorrhagic disease of infants

We studied molecular epidemiology of highly virulent echovirus 11 and 19 strains that were isolated during five outbreaks of enterovirus uveitis (EU) in Siberia in 1980-1989, and three outbreaks of multisystem hemorrhagic disease of infants (MHD) in 1988-1991. Three genome regions, 5'NTR, VP1-2A junction, and a fragment of 3D polymerase, were analyzed. Phylogenetic grouping in the VP1-2A region correlated with serotyping results. All studied EV11 and EV19 strains, including the prototype EV11 and EV19, formed a major phylogenetic group in VP1-2A region. Within that group, several EV11 isolates from EU and MHD outbreaks formed a distinct cluster in VP1-2A and 5' NTR genome regions, designated EV11/B. All strains of this cluster possessed high virulence for monkeys compared with the prototype echoviruses. Subgrouping within this cluster correlated with year of virus isolation, not with the disease the viruses caused in infants (EU or MHD).     

Acute enterovirus uveitis in infants

Enterovirus uveitis (EU) is a new infant eye disease that was first detected and identified in Russia in 1980-1981. Three subtypes of human echoviruses (EV19K, EV11A, and EV11/B) caused 5 nosocomial outbreaks of EU in different Siberian cities and towns in 1980-1989, by affecting more than 750 children mainly below one year of age. Sporadic and focal EU cases (more than 200) were also retrospectively diagnosed in other regions of Russia and in different countries of the former Soviet Union. There were following clinical manifestations: common symptoms of the infection; acute uveitis (rapid focal iridic destruction, pupillary deformities, formation of membranes in the anterior chamber of the eye); and in 15-30% of cases severe complications, cataract, glaucoma, vision impairments. Uveitis strains EV19 and EV11 caused significant uveitis in primates after inoculation into the anterior chamber of the eye, as well as sepsis-like fatal disease with liver necrosis after venous infection. The uveitis strains are phylogenetically and pathogenetically close for primates to strains EV19 and EV11 isolated from young children with sepsis-like disease. The contents of this review have been published in the Reviews in Medical Virology, 2004, vol. 14, p. 241-254.     

Enterovirus uveitis

Enterovirus uveitis (EU) is a new infant eye disease that was first observed in 1980. Three distinct subtypes of human echoviruses, EV19/K, EV11/A and EV11/B, caused five hospital outbreaks of EU in different Siberian cities in 1980-1989, affecting approximately 750 children, predominantly below 1 year of age. Sporadic EU cases were also retrospectively diagnosed in other regions of Russia and in different countries of the Former Soviet Union. The illness was characterised by rapid iris destruction and severe complications, including cataract and glaucoma. The disease has been a subject of intensive studies and was reproduced in lower primates after intraocular inoculation of isolated enterovirus strains. Importantly, prototype EV11 and EV19 strains did not induce notable disease in experimental monkeys. Some of the EU-causing strains were shown to be similar phylogenetically and in their pathogenetic properties to the enterovirus strains associated with multisystem hemorrhagic disease of newborns. In this review we present a summary of the vast epidemiological, virological, clinical and experimental data on this new form of ophthalmic infection.     

Detection of non-polio enteroviruses in Hungary 2000–2008 and molecular epidemiology of enterovirus 71, coxsackievirus A16, and echovirus 30

Human enteroviruses are associated with various clinical syndromes from minor febrile illness to severe, potentially fatal conditions like aseptic meningitis, paralysis, myocarditis, and neonatal enteroviral sepsis. Between June 2000 and August 2008 echovirus (E) type 2, 4, 6, 7, 9, 11, 13, 25, 30, coxsackievirus (CV) -A16, -A19, -B5, and enterovirus 71 (EV71) were reported in Hungary. In this study, 29 previously enterovirus positive samples from 28 patients diagnosed with hand, foot and mouth disease, meningitis and encephalitis, were molecularly typed. The genetic relationships of identified serotypes CV-A16, EV71, and E30 were assessed by direct sequencing of genomic region encoding the capsid protein VP1. The sequences were compared to each other and sequences from other geographical regions possessed in Genbank. The phylogenetic analysis of CV-A16 revealed that the viruses were mostly of Far-Eastern or Asia-Pacific origin. Typing of EV71 showed that one virus from 2000 belonged to genotype C1 and five viruses observed in 2004 and 2005 were identified as genotype C4. The 11 echovirus 30 strains showed homology with those of neighbor European countries. The molecular examination of E30 revealed that three separate lineages circulated in 2000, 2001, and 2004–2006 in Hungary.     

Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9

Echovirus 18 (EV18) is one of the echovirus serotypes associated with human diseases and in particular aseptic meningitis. To facilitate studies of the molecular epidemiology of EV18 and the evolution of enteroviruses in general, the complete nucleotide (nt) sequence was determined for the echovirus 18 prototype strain (Metcalf, EV18M). Excluding the poly A sequence, the genome consists of 7410 nt divided into a 740 nt 5' untranslated region (5' UTR), a 6567 nt long open reading frame coding for a 2189 amino acid (aa) polyprotein and a 103 nt 3' UTR. Molecular analysis of the EV18M genome showed a typical enterovirus-like organization. Phylogenetic analysis of the structural and non-structural genes revealed a pattern of different relationships to other echo- and coxsackieviruses. Similarity analysis demonstrated that the Hill strain of echovirus 9 is most likely the result of a previous recombination event between ancestors of the echovirus 9 strain Barty (5' half of the genome) and EV18M (3' half). Using a maximum likelihood approach, the recombination point was mapped to the 2C gene.     

Genetic diversity of enterovirus 71 isolated from cases of hand,foot and mouth disease in Yokohama City between 1982 and 2000

Summary.  Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980’s and 1990’s. Six EV71 isolates in the early 1980’s in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970’s. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan. Received June 25, 2002; accepted September 16, 2002     

Molecular epidemiology of echovirus 30 in Europe: succession of dominant sublineages within a single major genotype

Summary.  The genetic relationships between 131 echovirus type 30 (E-30) field isolates were studied using phylogenetic analysis of three genomic intervals: VP4/VP2 (420 nt), the entire VP1 and VP1/2A (150 nt). The strains had been isolated between 1975–1998, in different European countries, and in Israel and Japan. The maximum genetic variation was 15.7% in the VP4/VP2 region, 21.3% across the VP1/2A junction and 16.7% in the VP1-gene. The clustering patterns were very similar in all three regions. Two distinct genotypes were observed among the European strains, one of which was prevailing, spanning most of the investigated period. The same genotype was previously described to be the most prevalent circulating lineage of E-30 in Northern America. Interestingly, the two other genotypes comprising the prototype strain Bastianni and the oldest European isolates circulating before 1976, respectively, had apparently disappeared. Furthermore, the oldest lineages of the prevailing genotype had likewise disappeared and the recently isolated strains in the prevailing genotype were genetically quite homogenous, even when isolated in geographic regions far apart. These results indicate that the genetic variability of echovirus 30 is significantly lower than that of other previously characterized enteroviruses. Furthermore, one single, major genotype showed epidemic spread across two continents. Interestingly, despite the low nucleotide variability, maximum amino acid sequence variability in VP1 was surprisingly high, 8.0%, suggesting possible antigenical differences. Received May 12, 1999 Accepted August 22, 2000     

Newcastle disease outbreaks in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005 were caused by viruses of the genotypes VIIb and VIId

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world, and infections with virulent NDV strains continue to cause disease outbreaks in poultry and wild birds. To assess the evolutionary characteristics of 28 NDV strains isolated from chickens in Kazakhstan and Kyrgyzstan during 1998, 2000, 2001, 2003, 2004, and 2005, we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47–421) was performed using sequences of Kazakhstanian and Kyrgyzstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis demonstrated that the 14 newly characterized strains from years 1998 to 2001 belong to the NDV genotype VIIb, whereas the 14 strains isolated during 2003–2005 were of genotype VIId. All strains possessed a virulent fusion protein cleavage site (R-R-Q-R/K-R-F) and had intracerebral pathogenicity indexes in day-old chickens that ranged from 1.05 to 1.87, both properties typical of NDV strains classified in the mesogenic or velogenic pathotype.     

Pathotypical and genotypical characterization of strains of <Emphasis Type="Italic">Newcastle disease virus</Emphasis> isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001

Summary.  Twenty-nine strains of Newcastle disease virus (NDV) isolated from outbreaks in chicken and goose flocks in several regions of China during 1985–2001 were characterized pathotypically and genotypically. All except one of these strains were velogenic. For genotyping, phylogenetic tree analysis (nt 47–420), restriction site mapping (nt 334–1682) and residue substitution analysis (residues 4–124) of the F gene were performed using sequences of our isolates and sequences of selected NDV strains from GenBank. The results revealed that most of these newly characterized strains belonged to six novel genetic groups that were designated as VIf, VIg, VIIc, VIId, VIIe and IX. The genotype IX viruses, to which the China challenge strain F48E8 used for vaccine evaluation belonged, were found only in China and still induced sporadic infections in certain areas. Isolates belonging to group VIf and VIg were distinct from previously reported members of genotype VI, with genetic distance from 2.5 to 12.1%. Subgenotype VIIc, VIId and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for disease outbreaks in chicken and goose flocks and circulated predominantly in southern China in recent years. Finally, cross-protective testing showed that specific-pathogen free (SPF) chickens vaccinated with La Sota vaccines can be fully protected against challenge by strains from genetic groups VIb, VIg, VIId and IX, indicating that the antigenic differences between strains of various genotypes are insufficient to change the cross-protection conferred by the commonly used vaccine. Received August 16, 2002; accepted January 6, 2003 Published online April 28, 2003     

A large Finnish echovirus 30 outbreak was preceded by silent circulation of the same genotype

An outbreak of echovirus 30 (E-30) in 2009 was confirmed by both frequent isolation of the virus from sewage as well as from patient samples in Finland. Over the last 10 years E-30 had only been isolated sporadically in Finland. We here study the phylogenetic relationships of the strains from the outbreak in the context of E-30 circulation over the last 20 years. The analyzed region comprised 276 nucleotides in the 5′ end of VP1 (nucleotides 132–407 in the VP1 of the E-30 Bastianni strain). The Finnish strains were clustered into at least four distinct genogroups, with seven clusters exceeding the genotype demarcation of 12% and the 2009 epidemic strains forming the largest genogroup VII. Moreover, we detected largely divergent genotypes in 2007 and 2009. Interestingly, close genetic relatives of the epidemic strains had already been isolated a few years before the outbreak. Phylodynamic analysis estimated 8.9 years (95% highest posterior density intervals 7.0–11.0) as the age of genogroup VII, indicating a probable origin and evolutionary history prior to its introduction and epidemic expansion in Finland. Finally, the most recent common ancestor for the current E-30 diversity dates back to 1939 (95% highest posterior density intervals 1913–1956).     

Emergence of enterovirus 71 “double-recombinant” strains belonging to a novel genotype D originating from southern China: first evidence for combination of intratypic and intertypic recombination events in EV71

Hand–foot–mouth disease due to enterovirus 71 (EV71) and coxsackievirus A16 (CA16) has recently caused large outbreaks in mainland China in 2008. We performed complete genome sequencing on two EV71 (SZ/HK08-5 and SZ/HK08-6) and two CA16 (SZ/HK08-3 and SZ/HK08-7) strains from patients in Shenzhen, China. Phylogenetic, similarity plot and bootscan analyses revealed recombination between EV71 genotypes B and C at the 2A–2B junction, and between EV71 genotype B and CA16 strain G-10 in the 3C region for EV71 strains. A similar phenomenon was also found upon further gene sequencing with other EV71 strains. Recombination between CA16 strain G-10 and EV71 genotype A at the 2A–2B junction was also observed for CA16 strains. The present “double-recombinant” EV71 strains circulating in China and other EV71 subgenotype “C4” strains represent an additional genotype, D. CA16 strains should also be classified into two genotypes. This represents the first evidence for a combination of intratypic and intertypic recombination in EV71 strains.     

Competition binding studies with biotinylated echovirus 11 in cytofluorimetry analysis.

Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.     

Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008-2011

Hand, foot and mouth disease (HFMD) has mostly been caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). CA 16 was the most common cause of HFMD in 2010. EV71 had a high prevalence in 2008-2009 and has been identified with a higher frequency since 2011. Nearly complete genome sequences of three EV71 strains (2008-2009 strains) and two CA16 strains (2010 strains) obtained from outbreaks in Thailand in 2008 to 2010 were characterized. Based on a phylogenetic tree of the complete VP1 region, three EV71 strains grouped into the B5, C1 and C4 genotypes, and two CA16 strains grouped into the C genotype. Based on sequence analysis, nucleotide changes were found to cluster in the internal ribosome entry site (IRES) element of the 5′-untranslated region (5′-UTR). Amino acid differences identified in all strains were located in the non-structural protein. These data also provide the molecular epidemiology of EV71 and CA16 outbreaks in Thailand.     

Nucleotide sequences and mutations of the 5′-nontranslated region (5′NTR) of natural isolates of an epidemic echovirus 11′ (prime)

Summary.  An echovirus 11′ (prime) virus caused an epidemic in Hungary in 1989. The leading clinical form of the diseases was myocarditis. Hemorrhagic hepatitis syndroms were also caused, however, with lethal outcome in 13 new-born babies. Altogether 386 children suffered from registered clinical disease. No accumulation of serous meningitis cases and intrauterine death were observed during the epidemic, and the monovalent oral poliovirus vaccination campaign has prevented the further circulation of the virus. The 5′-nontranslated region (5′-NTR) of 12 natural isolates were sequenced (nucleotides: 260–577). The 5′-NTR was found to be different from that of the prototype Gregory strain (X80059) of EV11 (less than 90% identity), but related to the swine vesicular disease virus (D16364) SVDV and EV9 (X92886) as indicated by the best fitting dendogram. The examination of the variable nucleotides in the internal ribosomal entry site (IRES) revealed, that the nucleotide sequence of a region of the epidemic 5′-NTR was identical to that of coxsackievirus B2. Five of the epidemic isolates were found to carry mutations. Seven EV11′ IRES elements possessed identical sequences indicating, that the virus has evolved before its arrival to Hungary. The comparative examination of the suboptimal secondary structures revealed, that no one of the mutations affected the secondary structure of stem-loop structures IV and V in the IRES elements. Although it has been shown previously, that the echovirus group is genetically coherent and related to coxsackie B viruses the sequence differences in the epidemic isolates resulted in profound modification of the central stem (residues 477–529) of stem-loop structure No.V known to be affecting neurovirulence of polioviruses. Two alternate cloverleaf (stem-loop) structures were also recognised (nucleotides 376 to 460 and 540 to 565) which seem to mask both regions of the IRES element complementary to the 3′-end of the 18 S rRNA (460 to 466 and 561 to 570), thus probably diminishing initiation of translation. The possible biological importance of the alternative cloverleaf structures is supported by the fact that neither the 17 variable nucleotides nor the two mutations of epidemic isolates within the regions seem to modify the predicted alternative secondary structures in EV11, SVDV and CBV1-4. Accepted May 31, 2000 Received July 21, 1999     

Neutralization of different echovirus serotypes by individual lots of intravenous immunoglobulin

Replacement therapy using intravenous immunoglobulin (IVIG) preparations in people with antibody deficiencies is effective in preventing the majority of common bacterial and viral infections, yet echovirus break‐through infections have occurred. Currently, only limited information on neutralization capacity variability of individual IVIG lots against the different echovirus serotypes is available. Infectivity assays were established for the most prevalent echovirus serotypes (E 9, E 11, E 13, and E 30) circulating in the United States (US) and the European Union (EU). The echovirus serotype‐specific neutralization titers of 41 IVIG lots manufactured from either whole blood (Recovered) or collected by apheresis (Source) and from either the US or EU, were determined. Significantly higher (P < 0.0001) neutralization titers against E 11 and E 30 were found in IVIG lots manufactured from US Source plasma compared to US Recovered plasma. Geographically, IVIG lots made from US plasma contained significantly (P < 0.0001) higher neutralization titers against E 9 and E 11 than lots manufactured from EU plasma, whereas lots made from EU plasma showed significantly higher neutralization of E 30. To conclude, IVIG lots differ in their neutralizing antibody content against different echovirus serotypes, depending on plasma collection practices and geographic origin. Based on these results, an informed choice in selecting IVIG lots with the highest available neutralization titer against the specific echovirus serotype would seem to be beneficial during treatment of break‐through infections. J. Med. Virol. 83:305–310, 2011. © 2010 Wiley‐Liss, Inc.     

Hand, foot and mouth disease: seroprevalence of Coxsackie A16 and Enterovirus 71 in Germany

Coxsackie A16 (CA16) and Enterovirus 71 (EV71) are members of the picornaviridae family and are associated with hand, foot and mouth disease (HFMD), in rare cases also to acute neurological diseases. HFMD outbreaks have been reported from many parts of the world, especially Southeast Asia. The objective of the study was to analyze CA16 and EV71 seroepidemiologically in the population of Frankfurt/M., Germany. A total of 696 individuals (349 males and 347 females, divided into seven different age groups, 1–4, 5–9, 10–14, 15–19, 20–39, 40–59 and >60 years) were tested for serum antibodies against CA16 and EV71 by the use of a microneutralization test. Sera were collected at the Frankfurt university hospital from patients suffering from other diseases between March and September 2006. CA16 and EV71 infections were observed to be widely present in the population. The age-adjusted seroprevalence for individuals ≥1 year was found to be 62.9% for CA16 and 42.8% for EV71 without a gender-specific significant difference. Only 12.0 and 27.0% of the children aged 1–4 had antibodies to EV71 and CA16, respectively – indicating that 88 and 73% of the children in this age group were susceptible to the infection. A total of 213 individuals (30.6%) was seropositive for both viruses, 303 (43.5%) showed neutralizing antibodies (NtAb) to at least one of the two viruses. A total of 180 individuals (25.9%) revealed no antibodies. High CA16 and EV71 antibody titers were found especially in the age group of the 10- to 14-year-olds, without gender-specific difference. The seroprevalence study demonstrates a common spread of CA16 and EV71 in Germany, but a relatively high susceptibility of the younger population to CA16 and EV71. Obviously, the manifestation rate, i.e., distinct disease of these infections is low.     

Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus

Summary Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001–2004) were determined by sequencing open reading frames (ORFs) 2a–7. The ORFs 2a–7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296–11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France. The first two authors contributed equally to this work.     

Molecular phylogeny of VP1, 2A, and 2B genes of echovirus isolates: epidemiological linkage and observations on genetic variation

Summary. Phylogenetic relationships between 37 echovirus clinical isolates, most of them originating from an aseptic meningitis outbreak during 2001 in Greece, were investigated by RT-PCR and sequencing. The generic primers 292 and 222 were used to amplify about 300 bp of the 5′ end of VP1 while primers EUG3a, 3b, 3c, and EUC2 amplified the entire coding sequence of the 2A and 2B genes. Phylogenetic trees were constructed for each genomic region using the clinical isolates’ sequences and those of the prototype echoviruses in order to investigate the correlation of part of VP1 with the serotype as well as the genetic variation of the echovirus genome in 2A and 2B. The phylogenetic grouping pattern of the clinical isolates revealed that there is a correlation of serotype and genotype in the part of VP1 that was investigated, while this pattern is disrupted in the adjacent genomic regions that were sequenced. Sequence analysis of the adjacent 2A and 2B genes provided a different pattern of phylogenetic relationships and strong evidence of epidemiological linkage of most of the clinical isolates.     

Characterization of echovirus 22 variants

Summary Ten presumptive enterovirus isolates which could not be neutralized by type specific antisera to any prototype enterovirus were related to echovirus 22 using molecular, biologic and serologic methods. Viral protein fingerprinting and PCR first suggested that these strains were variants of echovirus 22. Three of the strains were echovirus 22 prime strains, i.e., antiserum made to the variant strain neutralized the variant and the prototype strain. The other strains were neutralized by antiserum to the prime strains. Unlike typical enteroviruses, echovirus 22 and 23 prototype viruses and 7 of the 10 variants were heat stable at 50 °C in H₂O for 1 h.     

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1

Summary.  Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia 1 virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I–IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 × 10⁻² synonymous substitutions per nucleotide per year. Received May 7, 2001 Accepted August 1, 2001