# Presentation: 090127

## Evolutionary Biology of Tospoviruses in Floral Crops

- J. W. Moyer
- North Carolina State University
- Department of Plant Pathology

- NORTH CAROLINA STATE UNIVERSITY

## Taxonomy

- TSWV is a member of the genus *Tospovirus* of the family *Bunyaviridae*. Other genera of *Bunyaviridae*:
*Bunyavirus*
*Phlebovirus*
*Hantavirus*
*Nairovirus*

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## Members of the Genus Tospovirus

- Members of the Genus *Tospovirus*

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## Genomic Organization

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## Virus Structure

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## Host Range 

- Wide host range: exceeds 900 plant species, spanning both monocotyledonous and dicotyledonous plant species
- Tremendous economic importance

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## Floral Crop Movement

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- Floral Crop Movement

## Genetics and evolution

- Heterogeneity and rapid adaptability are two prominent phenotypic characteristics that distinguish TSWV from many other plant viruses
- Existence of five strains of TSWV, separated from naturally occurring complexes (Norris, 1946)
- Six strains named as A, B, C1, C2, D, and E separated from tomato plants (Best & Gallus, 1953)
- First attempt to explain the “cross protection effect” with a new theory which implied transfer of character determinants from one virus particle to another (per se recombination)

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## Genetics and Evolution

- Mechanisms of evolution:
- Segment reassortment (Qiu, W.P., et al.)
- Mutation
- Defective interfering particles (DIs) (Resende, R.)
- Recombination ?
- Determinants of genetic structure:
- Genetic drift ?
- Selection ?

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## General Objectives

- Identify collaborators in the floral crop industry from diverse geographic regions, from which representative samples of TSWV and INSV populations can be obtained for characterization
- Characterize the diversity at the biological and molecular levels of representative populations

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## General Objectives

- Determine the changes in the natural populations due to changes in host and vectors
- Develop models of *Tospovirus* populations that can be used to identify the source of any given population: Attribution

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## Attribution

- The ability to target the source as well as to identify the causal pathogen

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## Objective 1

- Developed a network of collaborators: Argentina, Brazil, Colombia, Germany, Italy, Japan, Uruguay, and the USA including the industry trough AGDIA

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## Objective 2

- Technical steps toward analyses:
- Improved RNA extraction methods for floral hosts.
- PCR optimization for AU-rich regions of the genome (intergenic regions).
- Evaluation of primers for amplification and sequencing of the whole genome; S RNA (7), M RNA (12) and L RNA (18). Number of primer pairs in parentheses.
- Assembly and edition of nucleotide sequences with Vector NTi.
- Alignments of nucleotide sequences with CLUSTAL X.
- Edition of alignments with Genedoc 2.6.002
- Construction of haplotype maps with Paup, v 4.0 beta 4.
- Construction of Neighbor-joining phylograms with MEGA 2.1.
- Test of genetic differentiation between subpopulations with Permtest.
- Estimation of population genetics parameters, codon bias and examination of sequence divergence between species with DnaSP, v 3.51.
- Test of recombination with  LDhat.
- Identification of selection pressures per codon with the CODEML program of the PAML package, v 3.0d.

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## Diversity Between Populations

- S RNA:
- Direct sequencing of 13  isolates and subsequent analysis with data from the lab and GenBank.
- NSs : 21 sequences
- N : 41 sequences
- M RNA
- Direct sequencing of 13 isolates and subsequent analysis with data from the lab and GenBank.
- NSm: 17 sequences
- G1/G2: 19 sequences

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## S RNA

- CA

- NC

- SP

- BU

- NC

- NSs

- N

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## M RNA

**46.835**

**0.1449**

**0.0463**

**0.1185**

**0.035**

**G1/G2**

**43.102**

**0.1309**

**0.0361**

**0.1127**

**0.0303**

**NSm**

**46.654**

**0.1319**

**0.0427**

**0.0789**

**0.0235**

**N**

**46.666**

**0.0907**

**0.0373**

**0.0906**

**0.0341**

**NSs**

**ENC**

******(Sil)**

******(Tot)**

**Pi(Sil)**

**Pi(Tot)**

**Region**

- NSm

- G1/G2

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## Geographic Subdivision

| Region | Subpop. | Kst | Ks | P-value |
| --- | --- | --- | --- | --- |
| NSs | CA vs Bul. | 0.68889 | 0.010375 | 0.0* |
|  |  |  |  |  |
| N | Bul. vs GA | 0.507646 | 0.011668 | 0.0* |
|  | Bul. vs CA | 0.565285 | 0.00799 | 0.0* |
|  | CA vs GA | 0.456776 | 0.006781 | 0.0* |
|  |  |  |  |  |
| NSm | CA vs NC | 0.190789 | 0.017891 | 0.006* |
|  |  |  |  |  |
| G1/G2 | CA vs NC | 0.265368 | 0.018582 | 0.001* |

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## Intraspecific polymorphism

- High intraspecific polymorphism
- Estimation of the population parameters Pi(Sil), and (Sil), revealed a high polymorphism for each coding region, in agreement with the high mutation rates of RNA viruses.

**46.835**

**0.1449**

**0.0463**

**0.1185**

**0.035**

**G1/G2**

**43.102**

**0.1309**

**0.0361**

**0.1127**

**0.0303**

**NSm**

**46.654**

**0.1319**

**0.0427**

**0.0789**

**0.0235**

**N**

**46.666**

**0.0907**

**0.0373**

**0.0906**

**0.0341**

**NSs**

**ENC**

******(Sil)**

******(Tot)**

**Pi(Sil)**

**Pi(Tot)**

**Region**

**46.835**

**0.1449**

**0.0463**

**0.1185**

**0.035**

**G1/G2**

**43.102**

**0.1309**

**0.0361**

**0.1127**

**0.0303**

**NSm**

**46.654**

**0.1319**

**0.0427**

**0.0789**

**0.0235**

**N**

**46.666**

**0.0907**

**0.0373**

**0.0906**

**0.0341**

**NSs**

**ENC**

******(Sil)**

******(Tot)**

**Pi(Sil)**

**Pi(Tot)**

**Region**

| Region | Pi (Tot) | Pi (Sil) |  (Tot) |  (Sil) |
| --- | --- | --- | --- | --- |
| NSs | 0.03405 | 0.0906 | 0.0373 | 0.09069 |
| N | 0.02352 | 0.07885 | 0.04271 | 0.13188 |
|  |  |  |  |  |
| NSm | 0.03032 | 0.11272 | 0.03612 | 0.13085 |
| G1/G2 | 0.03499 | 0.11853 | 0.04626 | 0.14486 |

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##   Population Expansions→Geographic subdivision

| NSs
 | Pi(Tot)
 | Pi(Sil)
 | ∂(Tot)
 | ∂(Sil)
 | Tajimas
D | Fu & Li
D | Fu & Li
F |
| --- | --- | --- | --- | --- | --- | --- | --- |
| California | 0.006 | 0.016 | 0.0064 | 0.0193 | -0.4955 | -0.7047 | -0.725 |
| Bulgaria | 0.0166 | 0.034 | 0.0185 | 0.0404 | -0.532 | -0.734 | -0.808 |
|  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |
| N
 | Pi(Tot)
 | Pi(Sil)
 | ∂(Tot)
 | ∂(Sil)
 | Tajimas
D | Fu & Li
D | Fu & Li
F |
| California | 0.0022 | 0.0085 | 0.0032 | 0.0121 | -1.524* | -1.61* | -1.73* |
| Bulgaria | 0.0131 | 0.029 | 0.0189 | 0.0411 | -1.645* | -1.779* | -1.95* |
| Georgia | 0.0102 | 0.03939 | 0.0128 | 0.0478 | -1.0002 | -1.116 | -1.22 |
|  |  |  |  |  |  |  |  |

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## Test for recombination

- We used LDhat, which implements a coalescent-based method to detect and estimate recombination from gene sequences
- The null hypothesis (no recombination) is rejected if the proportion (PLPT) is less than a significance level (<0.05)
- NO EVIDENCE FOR RECOMBINATION FOR OUR DATA.

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## Interspecific Divergence

- Neutral theory predicts that the ratio of silent to replacement substitutions should be the same for polymorphisms within species and fixed differences between species.

| Region | FS | FR | PS | PR  | G value |
| --- | --- | --- | --- | --- | --- |
| NSs | 221 | 266 | 101 | 63 | 0.00032* |
| N | 126 | 165 | 94 | 40 | 0.0* |
|  |  |  |  |  |  |
| NSm | 123 | 132 | 88 | 23 | 0.0* |
| G1/G2 | 489 | 500 | 377 | 130 | 0.0* |

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## Selection

| Region | M2 vs M3 | M7 vs M8 | Pos. sites | dN/dS |
| --- | --- | --- | --- | --- |
| NSs | 14.6632* | 15.228*** | 458, 460 | 0.3671 |
| N | 8.092* | 11.586** | 10,174,255 | 0.2207 |
|  |  |  |  |  |
| NSm | 0.002 | 0.598 | ------------- | 0.0811 |
| G1/G2 | 1.15 | 6.364* | 530 | 0.1326 |

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## Conclusions

- TSWV has a strong spatial structure, attributed to founder effects:
- Significant genetic differentiation between subpopulations
- Decrease of genetic variation within subpopulations and
- Emergence of singletons in most of the analyzed loci
- High intraspecific polymorphism
- No evidence for recombination for the data analyzed

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## Objective 2-Future

- Define the structure of a single TSWV population (individual isolate): diversity within a population.
- Virus sources: plants from field and greenhouse operations from:
- Europe (Spain, Italy, Greece)
- South Africa
- Japan
- U.S.A.

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## Objective 2- Future

-       **Source**

**     ****Total RNA**

** ****cDNA**** **

**  ****PCR or**
** ****/ nested PCR**

-   **Cloning**

-   RFLP

-   **Sequencing**

- PCR of individual
- clones with inserts ( GC        clamp in 5’ end of forward primer)

- DGGE

- Heteroduplex analysis
- Heteroduples analysis-SSCP

- Denaturation

- Assymmetric PCR

-  PCR with one primer

- SSCP

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## Objective 3

- After defining the complexity of the structure certain selection pressures will be imposed:
- Mechanical passages
- Vector species
- Host differentials

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## Objective 3

- Found middle (M) RNA of TSWV is responsible for thrips transmission by a novel viral genetic system
- Analyzed M RNA sequences of non-transmissible and transmissible isolates
- Found a nucleotide deletion in glycoprotein coding region of non-transmissible isolates and confirmed glycoprotein may play an important role in thrips transmission
- Determined mutation frequencies of TSWV isolates maintained by repeated mechanical or thrips transmission
- Found specificity of vectors may map with M RNA of TSWV

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## Progression of thrips transmission rate of TSWV per mechanical passage (Mc Nulty, 2001; unpublished data).

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## Isolation of non-transmissible isolates from population A

-         Pop. A
- (mechanically-maintained, low transmissibility)

**T+**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T-**

**T+**

- Single Lesion Isolation

- Transmission Assay

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## Results

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- Isolation and identification of thrips non-transmissible and transmissible isolates from mechanically maintained RG2.

| Single lesion isolates | Transmission (infected/inoculated)1 | Transmissibility |
| --- | --- | --- |
| 2 | 0/30 ; 0/30 | -2 |
| 11 | 0/30 ; 0/30 | - |
| 28 | 0/30 ; 0/30 | - |
| 37 | 9/30 ; 8/30 | + |
| 41 | 7/30 ; 6/30 | + |
| 43 | 6/30 ; 7/30 | + |
| 81 | 0/30 ; 0/30 | - |

- 1. Transmission was confirmed by symptom development and RT-PCR-RFLP.  Individual experiments are separated
-     by semicolon.
- 2. + means transmissible and – means non-transmissible.

## Objective 4

- Extend the Attribution studies to include INSV.
- Other tospoviruses?
- Other viruses?
- Study the impact of host range and vector species in individual populations.
- Molecular and biological dissections of                                       individual populations.
- Partnership with AGDIA for industrial application.

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##    Sebastian Guyader

**     ****James Moyer**
**     ****Jorge Abad**
**Jan Speck**
**S.-H. Sin**
**Stephen New**

**Purugganan**
**Amy Lawton-Rauh**

-    **Sebastian Guyader**

**William Atchley**
**Michael J. Buck **

**Acknowledgements**

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