Department of Zoology, Tinbergen Building, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
Correspondence
Edward C. Holmes
Edward.Holmes{at}zoo.ox.ac.uk
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ABSTRACT |
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INTRODUCTION |
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One well-documented set of trade-offs in RNA virus evolution involves the vector-borne viruses of animals. In particular, evolutionary rates appear to be reduced in these viruses because of fitness trade-offs implicit when replicating in phylogenetically distinct species (vertebrates and invertebrates) that possess very different cell types (Scott et al., 1994; Weaver et al., 1999
). Most broadly, the limited range of insect vectors employed by viruses indicates that their evolution is strongly constrained by the requirements of vector transmission (Power, 2000
), and in some cases large-scale phylogenetic patterns appear to be governed by vector-type (Gaunt et al., 2001
). The constraints imposed on vector-borne RNA viruses are also apparent from molecular evolutionary studies. Jenkins et al. (2002)
recorded significantly lower rates of nucleotide substitution in RNA viruses transmitted by arthropod vectors than those viruses transmitted by other routes, apparently caused by increased purifying selection against amino acid changes in the former. In addition, weaker positive selection pressure was reported in the surface genes (mainly glycoproteins) of vector-borne viruses, suggesting that there are limitations to the number of available adaptive solutions (Woelk & Holmes, 2002
). Finally, an extremely strong force of purifying selection has been observed in the vector-borne dengue virus, where at least 90 % of the amino acid changes that arise at the intra-host level are deleterious in the long-term (Holmes, 2003b
).
Despite the growing evidence for complex fitness trade-offs in vector-borne RNA virus evolution, especially in patterns of nucleotide substitution, some experimental studies have shown that the replication of viruses in diverse cellular environments does not necessarily limit fitness increases. For example, Novella et al. (1999) showed that the alternate replication of Vesicular stomatitis virus (VSV) in insect and mammalian cells did not reduce the rate at which mutations appeared or inhibit fitness, as has also been observed in Eastern encephalitis virus (Weaver et al., 1999
). Similarly, Turner & Elena (2000)
evolved VSV on HeLa and MDCK cells and found that those viruses evolved in fluctuating environments performed as well as those adapted to a single environment. In general, in vitro studies of RNA virus evolution suggest that although adaptation to one host does indeed hinder the ability to adapt to new hosts, no loss of fitness occurs in viruses that are continually exposed to fluctuating environments (Elena, 2002
).
As a further test of the theory that replication in diverse hosts entails more adaptive constraints, we analysed the patterns of sequence evolution in vector-borne and non-vector-borne plant viruses. Plant viruses represent an ideal opportunity to determine whether host generalist and specialist viruses evolve in different ways. In particular, the capsid proteins (CPs) of plant viruses play a central role in their ability to survive in both plant and insect environments, and are implicated in practically every aspect of viral multiplication and dissemination. For example, not only does the CP encapsidate and protect the RNA genome, it is involved in genome replication, viral movement between cells and evasion of the plant defence system (Callaway et al., 2001). Viral CPs are therefore invariably required for transmission in vector-borne plant viruses, which in plants most often involves aphids (Lazarowitz, 2001
).
There are four general modes of vector-borne transmission employed by plant viruses, all of which rely on CP function, but which differ in the intimacy of the virusvector relationship. First, non-persistent, stylet-borne viruses are transmitted as a result of specific interactions between the virus and the vector stylet. Second, viruses that employ semi-persistent, foregut-borne transmission take longer to be acquired, but are retained in the vector for longer periods than stylet-borne viruses. Both these modes of transmission are said to be non-circulative as viruses are not absorbed into the haemocoel in the gut of the vector. Third, persistent, circulative transmission requires the vector feeding for several minutes so that the virus is acquired and then ingested into the gut. Within the gut, the virus must transverse the gut cell wall into the haemocoel, where it circulates and then enters the salivary glands by crossing further cell barriers. Finally, the virus is secreted with the salivary contents when the insect feeds. The final mode of transmission employed by a small number of plant viruses is persistent and propagative. This circulative mode of transmission is the only case in which active virus replication occurs in the vector, although all plant viruses replicate in plant cells. In this mode of transmission, once the virus has established an infection within the vector species, it remains a vector for its entire life.
Irrespective of the mode of transmission, interactions between virus and vector are complex, so that transmission is more than simply mechanical (Gray & Banerjee, 1999). For example, the potyviruses, which are persistent, stylet-borne viruses, are dependent on a highly conserved DAG motif in the CP for successful transmission (Blanc et al., 1997
; Gal-On et al., 1992
). As a consequence of these complex virusvector interactions, we hypothesize that vector-borne plant viruses will be subject to the same complex fitness trade-offs that have been observed in the vector-borne viruses of animals, as amino acid mutations that increase fitness in the host may have antagonistic effects in the vector (and vice versa) (Woelk & Holmes, 2002
). Herein we test this hypothesis.
As the molecular evolution of plant viruses has been relatively understudied, it is also important to compare their evolutionary dynamics with those of animal RNA viruses (García-Arenal et al., 2001; Woelk & Holmes, 2002
). The most informative comparison in this instance is between the external structural proteins of plant and animal viruses. Since most plant RNA viruses are non-enveloped it is the external structural CP that is critical for transmission. In the case of Tomato spotted wilt virus (TSWV), the only plant virus in our analysis to possess an envelope, experimental studies indicate that this external protein is essential for successful transmission in the gut of thrips (Medeiros et al., 2000
; Bandla et al., 1998
). In contrast, most animal vector-borne RNA viruses are enveloped and thus it is the envelope proteins and not the capsid proteins that are most important in transmission. Furthermore, the majority of animal vector-borne RNA viruses engage in circulative transmission which, as noted previously, involves the crossing of several cell membranes and hence requires complex adaptations. Indeed, the successful dissemination of the animal virus in the vector rests on the fusogenic and cell-receptor recognition qualities of the surface envelope proteins, as has been well documented in such viruses as the alphaviruses, flaviviruses and rhabdoviruses (Sanz et al., 2003
; Jones et al., 2003
; Langevin et al., 2002
). Conversely, internal structural (nucleocapsid) proteins of enveloped vector-borne animal viruses are not usually associated with transmission, but rather encapsidate newly formed RNAs and adhere to the outer lipid envelope. Indeed, Woelk & Holmes (2002)
found no significant difference in mean selection pressure for the internal structural proteins of vector-borne and non-vector-borne animal viruses, further suggesting that internal structural proteins do not distinguish vector and non-vector-borne transmission.
Plant viruses are responsible for billions of dollars of economic loss every year (Gray & Banerjee, 1999). Moreover, modern agricultural techniques, such as monoculture, have contributed to an explosion of emerging plant viruses, notably the geminiviruses, closteroviruses and tospoviruses. Given the importance of plant viruses to agriculture, it is imperative to gain a greater understanding of their evolutionary dynamics and what, if anything, limits their diversification. To this end, we conducted a comprehensive analysis of the selection pressures acting on the CPs of 36 plant viruses using published sequence data. Our study quantitatively tests whether the need for some plant viruses to survive in both plant and vector environments imposes increased selective constraints against amino acid change.
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METHODS |
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We employed an ML method to determine the selection pressures acting on the CP genes. The method uses different models of codon evolution to estimate the number of nonsynonymous (dN) and synonymous substitutions (dS) per site among codons, whilst accounting for the phylogenetic relationships among the sequences, thereby removing pseudo-replication (Yang et al., 2000). We compared the neutral M7 and the selection M8 models of codon evolution. Both models assume that the sequence alignment can be divided into 10 or 11 categories of dN/dS, with the dN/dS value of each category estimated from the data. In the case of M7, the dN/dS value for each of the 10 codon categories is constrained to be <1 so that positive selection is prevented. In contrast, M8 allows an eleventh category of codons where dN/dS can take on any value, including dN/dS >1, so that positive selection is allowed if this is a better fit to the data in hand. The support for these models is assessed using a likelihood ratio test with two degrees of freedom. Cases where M8 was significantly favoured over M7 and contains a category of codons with dN/dS >1 were taken as evidence for positive selection. Mean dN/dS ratios for each virus were estimated as the mean over all codons under M8 and were used to compare different groups of viruses (vector-borne and non-vector-borne viruses). All analyses were undertaken using the CODEML program from the PAML package (Yang, 1997
). A non-parametric statistical test (MannWitney U-test) was used to test for differences in selection pressure between virus groups since there is no basis to assume that dN/dS ratios for any group are normally distributed. Unfortunately, viral gene sequences from different families are so divergent that the effect of shared phylogenetic history on dN/dS cannot be assessed (Harvey & Pagel, 1993
).
Finally, to assess the overall extent of genetic diversity in each of our 36 datasets we measured the mean pairwise genetic distance (). These calculations were made using uncorrected distances (p-distances) with the MEGA2 package (Kumar et al., 2001
). As above, a MannWitney U-test was used to test for a difference in genetic diversity between vector-borne and non-vector-borne viruses.
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RESULTS |
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Of the 26 vector-borne viruses, only two were circulative. The enveloped circulative plant virus TSWV had a relatively high mean dN/dS ratio of 0·153, whereas the dN/dS ratio of the non-enveloped circulative plant virus, Rice black streaked dwarf virus was only 0·071. However, too few circulative plant viruses were available to conduct a meaningful comparison.
The number of host plant families a specific virus infected, varied between 1 (Potato virus A, Rice yellow mottle virus and Rice black streaked dwarf virus) and 32 (Turnip yellow virus), with a median number of seven families infected. Importantly, there was no statistical evidence to suggest that the number of plant families infected varies with the mean dN/dS of the virus (P=0·879, under the Spearman's rank correlation coefficient).
Comparing selective constraints among plant and animal viruses
We also compared the dN/dS ratios estimated here for the CP genes of plant RNA viruses with previously existing data for the surface structural proteins of vector and non-vector-borne animal viruses (Woelk & Holmes, 2002). The mean dN/dS ratios in the 25 non-enveloped vector-borne plant viruses were significantly greater than those in 18 vector-borne animal viruses; median (of mean) dN/dS ratios of 0·102 and 0·066, respectively, P=0·033 under a MannWhitney U-test. This indicates that plant viruses are subject to relatively weaker selective constraints compared with their animal counterparts. Conversely, dN/dS ratios in the 10 non-vector-borne plant viruses and 20 non-vector-borne animal RNA viruses were not significantly different; median (of mean) dN/dS ratios of 0·191 and 0·134, respectively, P=0·455 under a MannWhitney U-test.
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DISCUSSION |
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The finding that vector-borne plant RNA viruses are subject to greater constraint than those viruses not transmitted by vectors is intriguing, as the majority of the vector-borne viruses analysed here are non-circulative and therefore do not actively replicate in their insect hosts. Despite this, there is a wealth of data to support the theory that non-circulative plant viruses are involved in very specific interactions with their vectors (Power, 2000; Gray & Banerjee, 1999
). In particular, experimental studies have demonstrated the importance of specific domains and amino acids of the viral CP in transmissibility and vector specificity. Examples of plant virus genera where such complex interactions have been reported include Potyvirus (Atreya et al., 1991
, 1995
; Gal-On et al., 1992
), Luteovirus (Tamada et al., 1996
), Polerovirus (Brault et al., 1995
, 2003
), Enamovirus (Demler et al., 1997
) and Cucumovirus (Perry et al., 1994
, 1998
; Shintaku et al., 1992
; Liu et al., 2002
). Furthermore, the plant infecting umbraviruses, which lack a CP, are transmitted by mechanical inoculation and require encapsidation by luteoviruses to enable aphid transmission (Syller, 2002
). Finally, the observations that plant viral genera always utilize a single mode of vector transmission (either non-persistent non-circulative, semi-persistent non-circulative, persistent circulative or persistent propagative) and that each virus is faithful to a particular family of insects, lends further support to the idea that the vector acts as a major constraint in plant virus evolution and that new modes of transmission are difficult to evolve (Power, 2000
; Holmes, 2003a
).
In contrast to the association between transmission mode and selection pressure, we found no correlation between the number of plant families a virus infects and selection pressure in the CP. Indeed, many viruses infect a wide range of plant species. Hence, viruses are often able to infect different plant families with relative ease, so that the CP (or perhaps any viral protein) is not subject to particularly strong selective constraints from the plant. This interpretation is supported by a quantitative comparison of over 400 vector-borne plant viruses, which revealed that virus distribution is more constrained by virusvector than virusplant relationships (Power & Flecker, 2003). In summary, the selection imposed by a requirement for efficient vectors may be more severe than that set by plant defences (Power, 2000
).
Given the strong affects of vector apparent in our comparative sequence analysis, it becomes important to explain why viruses adapted to two environments are no less fit than those adapted to a single environment in experimental studies. The most likely explanation is that although extremely valuable, in vitro studies cannot fully capture the nature of long-term viral evolution in vivo. In particular, for vector-borne viruses the constraints associated with surviving the insect environment do not simply involve the ability to replicate efficiently in specific cell types. Rather, these viruses must also cross multiple cell membranes within the vector and contend with both vector- and host-immune systems. Hence, vector-borne RNA viruses in nature face far more complex environmental challenges than can be produced in cell culture. Moreover, although experimental studies show that viruses subject to fluctuating environments are equally as fit as those viruses evolving in a single environment, viruses passaged in a single cell type commonly experience fitness declines in the bypassed cells, revealing that mutations which are deleterious for alternative hosts do accumulate (Novella et al., 1999; Weaver et al., 1999
; Turner & Elena, 2000
; Elena, 2002
).
Comparative selection pressures in plant and animal RNA viruses
A key finding of our study was the observation that the CPs of vector-borne plant viruses were under significantly weaker selective constraint than the surface structural genes of vector-borne animal viruses. At first glance, these results seem puzzling as we might expect vector-borne plant viruses to be under greater constraint than vector-borne animal viruses, since the former must contend with phylogenetically more disparate environments (plant and insect) than the latter (vertebrate and insect). As this is not the case, it highlights differences in the virusvector interaction. Critically, all the (enveloped) vector-borne animal viruses studied were circulative, whereas all but one of the non-enveloped plant viruses were non-circulative. The circulative nature of the animal viruses necessitates their transport across several cell membranes, such that transport into the haemocoel and the salivary system of the insect is achieved (Gray & Banerjee, 1999). Conversely, non-circulative plant viruses are only required to bind to the surface of insect cells, necessitating a less complicated set of interactions. We propose that the greater complexity of interaction required for transmission of circulative viruses is reflected in a significantly stronger force of purifying selection against amino acid changes. However, it is also clearly the case that more sequence data are required to compare the circulative viruses of plants and animals. In our analysis, sufficient sequence data was only available from one circulative plant virus, Rice black streaked dwarf virus. Interestingly, the dN/dS ratio recorded for this virus (0·071) was relatively low compared with the median for vector-borne plant viruses (0·102), and closer to that of circulative animal viruses (0·066).
In combination with the recent work of Woelk & Holmes (2002), our analysis reveals that vector-borne RNA viruses are subject to greater selective constraints than those viruses transmitted by other routes, and hence, despite their high mutation rates, fitness trade-offs are commonplace in RNA virus evolution.
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ACKNOWLEDGEMENTS |
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Received 25 March 2004;
accepted 23 June 2004.