Worldwide spread of Dengue virus type 1

BACKGROUND: DENV-1 is one of the four viral serotypes that causes Dengue, the most common mosquito-borne viral disease of humans. The prevalence of these viruses has grown in recent decades and is now present in more than 100 countries. Limited studies document the spread of DENV-1 over the world despite its importance for human health. METHODOLOGY/PRINCIPAL FINDINGS: We used representative DENV-1 envelope gene sequences to unravel the dynamics of viral diffusion under a Bayesian phylogeographic approach. Data included strains from 45 distinct geographic locations isolated from 1944 to 2009. The estimated mean rate of nucleotide substitution was 6.56 × 10⁻⁴ substitutions/site/year. The larger genotypes (I, IV and V) had a distinctive phylogenetic structure and since 1990 they experienced effective population size oscillations. Thailand and Indonesia represented the main sources of strains for neighboring countries. Besides, Asia broadcast lineages into the Americas and the Pacific region that diverged in isolation. Also, a transmission network analysis revealed the pivotal role of Indochina in the global diffusion of DENV-1 and of the Caribbean in the diffusion over the Americas. CONCLUSIONS/SIGNIFICANCE: The study summarizes the spatiotemporal DENV-1 worldwide spread that may help disease control.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.

Populationsdynamik des Cydia pomonella Granulovirus

Das Cydia pomonella Granulovirus (CpGV, Fam. Baculoviridae) ist ein sehr virulentes und hoch spezifisches Pathogen des Apfelwicklers (Cydia pomonella), das seit mehreren Jahren in der Bundesrepublik Deutschland und anderen Ländern der EU als Insektizid zugelassen ist. Wie andere Baculoviren auch befällt es die Larven der Insekten und ist aufgrund seiner Selektivität für Nicht-Zielorganismen unbedenklich. In der Vergangenheit konzentrierte sich die Erforschung des CpGV auf Bereiche, die für die Anwendung im Pflanzenschutz relevant waren, wobei nach fast 20 Jahren nach der ersten Zulassung noch immer nicht bekannt ist, ob und wie sich das CpGV in der Umwelt etablieren kann. Im Rahmen der vorliegenden Arbeit wurden verschiedene Parameter, mit deren Hilfe die Populationsdynamik des CpGV beschrieben werden kann, analysiert und quantitativ bestimmt. Neben den biologischen Eigenschaften wie Virulenz, DNA-Charakterisierung und Quantifizierung der Virusnachkommenschaft wurden insbesondere die horizontale sowie die vertikale Transmission, die Inaktivierung und die Infektion später Larvenstadien untersucht. Letztlich wurden die ermittelten Parameter zusammen mit Daten aus der Literatur in ein mathematisches Modell integriert. Um die Wahrscheinlichkeit der horizontalen Transmission zu quantifizieren, wurde ein Modellsystem mit losen Äpfeln etabliert, in dem verschiedene Szenarien möglicher horizontaler Transmission unter definierten Laborbedingungen getestet wurden. In Versuchsserien, in denen ein Virusfleck, entsprechend der produzierten Virusmenge einer Eilarve, auf einen Apfel appliziert worden war, war unter den aufgesetzten Apfelwicklerlarven lediglich eine sehr geringe Mortalität von 3 - 6% zu beobachten. Wurde jedoch ein an einer Virusinfektion gestorbener Larvenkadaver als Inokulum verwendet, lag die Mortalitätsrate aufgesetzter Larven bei über 40%. Diese beobachtete hohe horizontale Transmissionsrate konnte mit dem Verhalten der Larven erklärt werden. Die Larven zeigten eine deutliche Einbohrpräferenz für den Stielansatz bzw. den Kelch, wodurch die Wahrscheinlichkeit des Zusammentreffens einer an der Infektion verendeten Larve mit einer gesunden Larve um ein Vielfaches zunahm. In einem ähnlich angelegten Freilandversuch konnte eine horizontale Transmission nicht belegt werden. Der Unterschied zur Kontrollgruppe fiel aufgrund einer hohen natürlichen Mortalität und einer damit einhergehenden niedrigen Dichte der Larven zu gering aus. Parallel hierzu wurde außerdem eine Halbwertszeit von 52 Sonnenstunden für das CpGV ermittelt. Weiterhin konnte festgestellt werden, dass die Mortalität von späteren Larvenstadien, die 14 Tage Zeit hatten sich in die Äpfel einzubohren, bevor eine CpGV-Applikation durchgeführt wurde, ebenso hoch war wie bei Larven, die sich im L1-Stadium auf der Apfeloberfläche infizierten. Aufgrund des höheren Alters jener Larven war der Fraßschaden an befallenen Äpfeln jedoch wesentlich größer und vergleichbar mit dem Fraßschaden einer unbehandelten Kontrolle. Der Versuch zur vertikalen Transmission zeigte dass, obwohl die verwendete Apfelwicklerzucht nicht frei von CpGV war, die Mortalitätsrate der Nachkommen subletal infizierter Weibchen (44%) jedoch deutlich höher war als die der Nachkommen subletal infizierter Männchen (28%) und der unbehandelten Kontrolle (27%). Auch in den PCR-Analysen konnte eine größere Menge an CpGV-Trägern bei den Nachkommen subletal infizierter Weibchen (67%) als bei den Nachkommen subletal infizierter Männchen (49%) und bei der Kontrolle (42%) nachgewiesen werden. Die Ergebnisse deuten darauf hin, dass eine Infektion durch subletal infizierte Weibchen vertikal in die nächste Generation übertragen werden kann. Dies lässt erkennen, dass in der Folgegeneration des Apfelwicklers eine zusätzliche Wirkung des CpGV durch vertikale Transmission auftreten kann. Hierin wäre auch ein potentieller Mechanismus für eine dauerhafte Etablierung des Virus zu sehen. Letztlich wurden alle Parameter, die die CpGV-Apfelwickler-Beziehung beschreiben, in ein mathematisches Modell GRANULO integriert. Nach einer Sensitivitätsanalyse wurde GRANULO teilweise mit Daten aus den Freilandversuchen verifiziert. Durch Modifikation der Virusparameter im Modell konnte anschließend der Einfluss veränderter biologischer Eigenschaften (UV-Stabilität und Transmissionsraten) der Viren in Simulationen theoretisch erprobt werden. Das beschriebene Modell, das allerdings noch einer weitergehenden Verifizierung und Validierung bedarf, ist eine erste Annäherung an die quantitative Erfassung und Modellierung der Populationsdynamik des Systems CpGV-Apfelwickler. Die im Zusammenhang mit der Populationsdynamik des Apfelwicklers erhobenen Daten können einen wertvollen Beitrag zur Optimierung von Kontrollstrategien des Apfelwicklers mittels CpGV leisten. Außerdem geben sie Aufschluss über die Etablierungsmöglichkeiten dieses Bioinsektizids.

Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing

A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended.

Virus a RNA e a DNA: importanza dell’ampliamento dello spettro d’ospite nell’evoluzione virale

L’ampliamento dello spettro d’ospite è strettamente connesso al processo evolutivo a cui i virus sono assoggettati e rappresenta una notevole sfida alla loro capacità di adattarsi. L’attitudine a superare le barriere di specie è conseguente alla costante e relativamente rapida evoluzione che caratterizza i virus; allo stesso tempo, la forza selettiva esercitata dal nuovo ospite rappresenterà un ulteriore stimolo per le capacità adattative del virus. Ad oggi, i meccanismi genetici ed evolutivi responsabili del salto di specie virale, cioè la trasmissione di un virus da un ospite tradizionale ad uno precedentemente resistente all’infezione, sono parzialmente sconosciuti. Nel seguente lavoro verranno presentati gli studi effettuati sulle dinamiche evolutive caratterizzanti virus a RNA e a DNA in cui si sono osservate variazioni dello spettro d’ospite. Gli studi hanno riguardato i coronavirus, con particolare riferimento al ruolo svolto dai pipistrelli nell’evoluzione dei coronavirus SARS-correlati, e l’importanza del gatto nell’evoluzione dei parvovirus dei carnivori. Nella prima sezione saranno mostrate le correlazioni genetiche dei coronavirus identificati in Italia nei pipistrelli appartenenti alla specie Rhinolophus ferrumequinum con i ceppi europei e del resto del mondo, allo scopo di chiarire l’origine evolutiva dei coronavirus dei pipistrelli correlati al virus della SARS (Bat-SARS-like CoV) europei, gli eventi migratori che hanno caratterizzato la loro diffusione nel continente e le potenziali ripercussioni sulla salute pubblica. Nella seconda sezione saranno evidenziate le caratteristiche molecolari dei ceppi di parvovirus circolanti nella popolazione felina, valutandone la diversità di sequenza e la complessità genetica, allo scopo di ottenere importanti informazioni in merito all’evoluzione del virus e alle interazioni tra il parvovirus e l’ospite.

Epidemiologia e caratterizzazione molecolare di virus emergenti responsabili di Zoonosi: Calcivirus e Virus dell'Epatite E

Il virus dell’Epatite E (HEV) e i calicivirus (norovirus e sapovirus) causano rispettivamente epatite acuta e gastroenterite. Questi virus sono considerati agenti eziologici emergenti rappresentando un problema di sanità pubblica e di sicurezza alimentare. Per HEV, è ormai confermata la trasmissione zoonotica, e il suino è considerato il principale serbatoio asintomatico. Norovirus e sapovirus infettano sia i bambini che gli adulti. Sebbene questi virus siano stati identificati anche negli animali, la possibile trasmissione zoonotica non è stata dimostrata in modo conclusivo. Il lavoro sperimentale condotto durante il Dottorato di Ricerca è stato focalizzato sullo studio degli aspetti biologici ed epidemiologici dell’infezioni causate da HEV e da calicivirus. Per la prima volta in Italia, i risultati ottenuti hanno dimostrato la presenza del virus HEV nei fegati di suini in fase di macellazione ed hanno confermato, attraverso la ricerca di anticorpi, un’elevata esposizione degli animali al virus. Inoltre, mediante la produzione di antigeni e reattivi immunologici, sono stati messi a punto test diagnostici per la ricerca di anticorpi contro HEV nel suino e nei cinghiali. Il lavoro svolto per la ricerca di calicivirus nel suino e nel bovino ha dimostrato la circolazione dei sapovirus in popolazioni di suini asintomatici e la presenza di norovirus nei vitelli affetti da diarrea acuta.Sono stati inoltre sviluppati reattivi immunologici, utilizzando proteine del capside di norovirus umano e bovino espresse con il sistema ricombinante baculovirus. Questi hanno permesso di evidenziare la presenza di anticorpi contro norovirus umano e bovino, in sieri di veterinari professionalmente esposti. Inoltre, sono stati utilizzati per sviluppare metodi per la concentrazione dei virus da matrici a bassa concentrazione.Infine, le VLP sono state utilizzate per valutare l’attivazione del sistema immunitario umano ex vivo. I risultati hanno dimostrato che le VLP di NoV stimolano il sistema immunitario attivando risposte di tipo Th1 e Th2 .

Sorveglianza dell’infezione da virus dell’epatite E (HEV) in Italia: from farm to table

L'epatite E è una malattia umana con caratteristiche di epatite acuta, causata da un ssRNA virus (HEV). Nel 1997, HEV è stato identificato per la prima volta nei suini (SwHEV). In seguito, diverse evidenze, tra cui la vicinanza genetica tra ceppi umani e suini, suggerirono la trasmissione zoonotica del virus. Nella presente tesi, l’identificazione di SwHEV è stata condotta mediante ricerca di porzioni di genoma virale attraverso RT-PCR. Dal 2011 al 2013, sono stati analizzati 343 campioni fecali (da 19 allevamenti) e 70 bili (da 2 macelli) prelevati da altrettanti suini, in diverse Regioni italiane. E’ stato inoltre condotto uno studio retrospettivo su 78 feci (da 3 allevamenti) raccolte nel 2000. Il virus è stato identificato nel 24,5% e 19,2% delle feci raccolte rispettivamente nel 2011-2013 e nel 2000. Nessuna bile è risultata positiva. Mediante sequenziamento del genoma intero di uno dei virus identificati, è stata condotta l’analisi filogenetica per valutarne il grado di correlazione con alti ceppi suini e umani. La presenza di HEV è stata valutata lungo la filiera di produzione suina, dal macello al punto vendita. Trentaquattro campioni di feci, fegato e muscolo sono stati raccolti in un macello da altrettanti suini sani (età:6-7 mesi). Quattordici feci e 2 fegati, sono risultati positivi per HEV. Sono state prelevate 129 salsicce sia allo stabilimento di trasformazione sia alla vendita, ma nessuna è risultata positiva. La presenza di HEV è stata valutata anche nelle salsicce di fegato, fresche e secche, acquistate presso una macelleria. Il genoma virale è stato rilevato nel 22,2% delle salsicce fresche e nel 4,3 % di quelle secche ma la vitalità del virus non è stata dimostrata. In conclusione, lo studio condotto ha confermato l’ampia circolazione di HEV nei suini e la possibile contaminazione dei prodotti carnei derivati, confermando la necessità di una continua sorveglianza.

Detection and molecular characterization of viruses infecting Actinidia spp.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

Phylogenetic approach reveals that virus genotype largely determines HIV set-point viral load

HIV virulence, i.e. the time of progression to AIDS, varies greatly among patients. As for other rapidly evolving pathogens of humans, it is difficult to know if this variance is controlled by the genotype of the host or that of the virus because the transmission chain is usually unknown. We apply the phylogenetic comparative approach (PCA) to estimate the heritability of a trait from one infection to the next, which indicates the control of the virus genotype over this trait. The idea is to use viral RNA sequences obtained from patients infected by HIV-1 subtype B to build a phylogeny, which approximately reflects the transmission chain. Heritability is measured statistically as the propensity for patients close in the phylogeny to exhibit similar infection trait values. The approach reveals that up to half of the variance in set-point viral load, a trait associated with virulence, can be heritable. Our estimate is significant and robust to noise in the phylogeny. We also check for the consistency of our approach by showing that a trait related to drug resistance is almost entirely heritable. Finally, we show the importance of taking into account the transmission chain when estimating correlations between infection traits. The fact that HIV virulence is, at least partially, heritable from one infection to the next has clinical and epidemiological implications. The difference between earlier studies and ours comes from the quality of our dataset and from the power of the PCA, which can be applied to large datasets and accounts for within-host evolution. The PCA opens new perspectives for approaches linking clinical data and evolutionary biology because it can be extended to study other traits or other infectious diseases.

Molecular epidemiology reveals long-term changes in HIV type 1 subtype B transmission in Switzerland

Sequence data from resistance testing offer unique opportunities to characterize the structure of human immunodeficiency virus (HIV) infection epidemics.

Novel hepatitis E virus genotype in Norway rats, Germany

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E–like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.

Combining spatial-temporal and phylogenetic analysis approaches for improved understanding on global H5N1 transmission

Background Since late 2003, the highly pathogenic influenza A H5N1 had initiated several outbreak waves that swept across the Eurasia and Africa continents. Getting prepared for reassortment or mutation of H5N1 viruses has become a global priority. Although the spreading mechanism of H5N1 has been studied from different perspectives, its main transmission agents and spread route problems remain unsolved. Methodology/Principal Findings Based on a compilation of the time and location of global H5N1 outbreaks from November 2003 to December 2006, we report an interdisciplinary effort that combines the geospatial informatics approach with a bioinformatics approach to form an improved understanding on the transmission mechanisms of H5N1 virus. Through a spherical coordinate based analysis, which is not conventionally done in geographical analyses, we reveal obvious spatial and temporal clusters of global H5N1 cases on different scales, which we consider to be associated with two different transmission modes of H5N1 viruses. Then through an interdisciplinary study of both geographic and phylogenetic analysis, we obtain a H5N1 spreading route map. Our results provide insight on competing hypotheses as to which avian hosts are responsible for the spread of H5N1. Conclusions/Significance We found that although South China and Southeast Asia may be the virus pool of avian flu, East Siberia may be the source of the H5N1 epidemic. The concentration of migratory birds from different places increases the possibility of gene mutation. Special attention should be paid to East Siberia, Middle Siberia and South China for improved surveillance of H5N1 viruses and monitoring of migratory birds.

Antiretroviral therapy for prevention of HIV transmission in HIV-discordant couples

Antiretroviral drugs have been shown to reduce risk of mother-to-child transmission of human immunodeficiency virus (HIV) and are also widely used for post-exposure prophylaxis for parenteral and sexual exposures. Observational data, ecological studies and models suggest that sexual transmission may be lower in couples in which one partner is infected with HIV and the other is not and the infected partner is on antiretroviral therapy (ART).

Antiretroviral therapy for prevention of HIV transmission in HIV-discordant couples

Antiretroviral drugs have been shown to reduce risk of mother-to-child transmission of human immunodeficiency virus (HIV) and are also widely used for post-exposure prophylaxis for parenteral and sexual exposures. Observational data, ecological studies and models suggest that sexual transmission may be lower in couples in which one partner is infected with HIV and the other is not and the infected partner is on antiretroviral therapy (ART).