Sequences of Citrus tristeza virus separated in time and space are essentially identical

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.     

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

Citrus tristeza virus(CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide.To develop reliable and effective serological detection assays of CTV,the major capsid protein(CP) gene of CTV was expressed in Escherichia coli BL21(DE3) using the expression vector p ET-28 a and purified through Ni~+-NTA affinity chromatography.The recombinant protein was used to immunize BALB/c mice.Four hybridoma cell lines(14B10,14H11,20D5,and20G12) secreting monoclonal antibodies(MAbs) against CTV were obtained through conventional hybridoma technology.The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10-7 in indirect enzyme-linked immunosorbent assay(ELISA).Western blots showed that all four MAbs could specifically react with CTV CP.Using the prepared MAbs,dot-ELISA,Tissue print-ELISA,and triple antibody sandwich(TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies.The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and1:10,240(w/v,g/m L),respectively.Tissue print-ELISA was particularly useful for large-scale field sample detection,mainly owing to its simplicity and lack of sample preparation requirements.The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality,Jiangxi Province,and Zhejiang Province of China.The coincidence rate of serological and RT-PCR test results reached more than 99.5%.The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts

Summary A strategy for the sereening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol-(PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check eitrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virusderived sequences and virus challenge of transgenic cells are presented.     

Citrus tristeza virus: survival at the edge of the movement continuum

Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a "pure" culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature.     

Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus

Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.     

Intra-farm diversity and evidence of genetic recombination of Citrus tristeza virus in Delhi region of India

Infection of Citrus tristeza virus (CTV) in different citrus orchards of New Delhi was detected by direct antigen coated-ELISA and RT-PCR. Sweet orange (Citrus sinensis) orchards were found to be susceptible to CTV with estimated disease incidence up to 39%. Kagzi kalan (C. lemon), Pumello (C. paradisi) and Kinnow mandarin (C. reticulata) orchards did not show CTV infection. Three CTV isolates, D1, D7 and D15 randomly selected from infected sweet orange orchards were considered for biological and molecular characterization. In the host range study, all the Delhi isolates infected Darjeeling mandarin (C. reticulata), Kagzi lime (C. aurantifolia), sour orange (C. aurantium) and sweet orange but not Kinnow mandarin. A fragment of 5??ORF1a and complete coat protein (CP) gene of these three isolates were cloned, sequenced and compared with other Indian and international CTV isolates. Delhi isolates shared 85?C92% sequence identity for 5??ORF1a fragment and 89?C91% for CP gene among them. Phylogenetic analysis segregated three Delhi isolates into three genogroups for each of 5??ORF1a fragment and CP gene, however phylogenetic relationships for both the genomic regions was incongruent. Recombination detecting program RDP3 detected CTV isolate D7 as recombinant, indicating genetic variability in CTV isolates might be the outcome of recombination events between divergent CTV sequences. An attempt was made in present study to characterize CTV isolates biologically and at genetic level, and to determine genetic diversity at farm level and study the recombination of CTV isolates in Delhi region.     

Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.     

Livestock production and animal health in Sicily, Italy

In Sicily, as in other Mediterranean areas, livestock represents one of the most important resources for the island economy. This sector involves more than 16,000 farms of cattle and 10,000 farms of sheep and goats (respectively 6% and 15% of national production) which are actually increasing their number. Most livestock in Sicily is owned by small holders and pastoralists. Regional production of milk feeds some industries, which involve a large and increasing occupational area. Due to its peculiar geographic aspect Sicily is constituted by hill areas with sporadic grasses, therefore extensive grazing methods represent an ancient, traditional practice for using poor lands. For the control of infectious diseases Veterinary Services (VS) are based on the public regional network that is coordinated by the Ministry of Health in Rome. Even if Sicilian VS had to solve many constraints related to traditional "pastoral management system", to the lack of any sanitary background of this sector, to unknown data on the numbers of the whole livestock, the effort done in the last years has allowed to monitor for brucellosis, bovine leukemia virus (BLV), bovine TBC, swine vescicular disease (MVS) and other diseases for the majority of the farms. Tick-borne diseases (TBDs) still represent a serious impediment to the improvement of meat and milk production in the region. Every year, several outbreaks related to Theileria, Babesia or Anaplasma infections in cattle are recorded. Imported breeds pay the most expensive tribute often with a mortality rate of 100%. In the last five years more than 170 outbreaks of TBDs have been notified by our Institute although the pathogen prevalence and economical impact in the Sicilian livestock is still unknown. The outdoor grazing of the animals, far away from observation, a subclinical course of the disease, can in fact create difficulties in discovering infection and therefore the therapy is often too late. New diagnostic and control methods (PCR, vaccination) are being developed at the Istituto Zooprofilattico Sperimentale of Sicily in cooperation with other national and international laboratories (such as the University of Utrecht).     

Phylodynamics of the Emergence of Influenza Viruses after Cross-Species Transmission

Human populations are constantly exposed to emerging pathogens such as influenza A viruses that result from cross-species transmissions. Generally these sporadic events are evolutionary dead-ends, but occasionally, viruses establish themselves in a new host that offers a novel genomic context to which the virus must adjust to avoid attenuation. However, the dynamics of this process are unknown. Here we present a novel method to characterize the time it takes to G+C composition at third codon positions (GC3 content) of influenza viruses to adjust to that of a new host. We compare the inferred dynamics in two subtypes, H1N1 and H3N2, based on complete genomes of viruses circulating in humans, swine and birds between 1900–2009. Our results suggest that both subtypes have the same fast-adjusting genes, which are not necessarily those with the highest absolute rates of evolution, but those with the most relaxed selective pressures. Our analyses reveal that NA and NS2 genes adjust the fastest to a new host and that selective pressures of H3N2 viruses are relaxed faster than for H1N1. The asymmetric nature of these processes suggests that viruses with the greatest adjustment potential to humans are coming from both birds and swine for H3N2, but only from birds for H1N1.     

Phylodynamics and human-mediated dispersal of a zoonotic virus

Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.     

Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot Hybridization

A non‐radioactive dot‐blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)‐labelled minus‐sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.     

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus (CTV,Closterovirus) in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years (2012 and 2013). The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real‐time RT‐PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.     

Evolution and Molecular Epidemiology of Citrus tristeza virus on Crete: Recent Introduction of a Severe Strain

Sporadic incidences of Citrus tristeza virus (CTV) in western Crete resulting from the introduction of a mild strain (Spanish isolate T385) have been reported previously. Further analysis within this region has identified an emerging second CTV strain with minimal genetic divergence, sharing 99% nucleotide identity with the severe stem‐pitting isolate Taiwan‐Pum/SP/T1. Other severe isolates from the Mediterranean region appear in the same phylogenetic cluster, indicating movement or new introductions and the need for targeted control actions and improved phytosanitary measures in this area.     

Clinical and laboratory studies on theileriosis outbreaks in Sicily, Italy

A total of 153 blood samples were collected all over the Sicilian island in 15 farms. 29.4% and 25.5% were PCR positive for Theileria annulata and T. buffeli/orientalis respectively. 15.7% of cattle blood samples showed a mixed infection with both Theileria species. Each farm showed an infection rate ranging from 0% to 100%. Hyalomma marginatum and Boophilus annulatus were the most common ticks collected in April and November 1998 respectively. The aim of the present work was to investigate on haemoparasite species spread in Sicily. Ticks vectors involved in the transmission, distribution, risk areas and seasons, state of healthy carrier, are also discussed in this paper.     

Variation of haplotype distributions of two genomic regions of Citrus tristeza virus populations from eastern Spain

Genetic variation in natural populations of Citrus tristeza virus (CTV) was studied using haplotypes detected by single-strand conformation polymorphism (SSCP) analysis of two genomic regions (p20 gene and segment A, located in ORF1a). Analysis of 254 samples from 125 trees, collected at 12 different sites, yielded 8 different haplotypes for p20 and 5 for segment A. The most frequent haplotype of p20 was predominant at all sites, but several sites differed in the predominance of segment A haplotypes. At most sites, the homozygosity observed for the p20 gene tended to be higher than expected in a neutral evolution, whereas the opposite was true for segment A. Comparison of the populations at different sites showed that 44 of the 66 possible population pairs were genetically distinct for segment A, but only six pairs differed for the p20 gene. Analysis of molecular variance grouping trees by site, scion variety, rootstock or age, showed that variation in segment A was significantly affected by site, tree age and rootstock, and that variation between trees in each group and within trees was even more important. In contrast, variation in p20 was affected only by site and rootstock, each factor contributing to < 2% of the variation. The data suggest that sequence variations in segment A must be functionally less important and that it has less evolutionary constraints than p20. Detection of different haplotypes in neighbour trees or in samples from the same tree may help explain part of the variability observed in CTV symptom expression.