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Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011-2012.

Rizzo F, Cerutti F, Ballardini M, Mosca A, Vitale N, Radaelli MC, Desiato R, Prearo M, Pautasso A, Casalone C, Acutis P, Peletto S, Mandola ML - Parasit Vectors (2014)

Bottom Line: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment.Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown.These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

View Article: PubMed Central - PubMed

Affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, I-10154 Torino, Italy. simone.peletto@izsto.it.

ABSTRACT

Background: The genus Flavivirus comprises several mosquito-borne species, including the zoonotic pathogens West Nile and Usutu virus, circulating in animals and humans in Italy since 1998. Due to its ecological and geographical features, Piedmont is considered a risk area for flavivirus transmission. Here we report the results of a flavivirus survey (detection and genetic characterization) of mosquitoes collected in Piedmont in 2012 and the genetic characterization of three strains detected in 2011.

Methods: Pools of 1-203 mosquitoes, upon RNA extraction with TRIzol, were screened by a PCR assay for a 263 bp fragment of the Flavivirus NS5 gene. All positive samples were tested with a specific PCR for the E protein gene of Usutu virus and a generic Flavivirus RT-nested-PCR for a larger tract of the NS5 gene before sequencing. Phylogenetic trees were built with both NS5 fragments of representative Flavivirus species. DNA extracts of part of the positive pools were tested to detect sequences integrated in the host genome.

Results: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment. Among the 19 sequences correlating with the insect-specific flavivirus group, ten samples, retrieved from Aedes albopictus, clustered within Aedes flavivirus, while the other nine aggregated in a separate clade composed of strains from various mosquito species (mainly Aedes vexans) from Piedmont and the Czech Republic. Six out of these nine also presented a DNA form of the sequence. The remaining sequences belonged to the mosquito-borne group: four, all from Culex pipiens, correlated to Italian Usutu virus strains, whereas two, from Ochlerotatus caspius, were highly similar to Marisma mosquito virus (MMV).

Conclusions: Our findings confirm the circulation of Usutu virus and of the potentially zoonotic Marisma mosquito virus in Piedmont. This is the first detection of Aedes flavivirus in Piedmont. Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown. These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

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Phylogenetic tree based on the long NS5 fragment of the Flavivirus genus. A fragment of 937 bp of the NS5 gene was used to infer the tree. The phylogenetic analysis includes 35 reference sequences from GenBank and 20 from this study. Unpublished sequences are given in bold, Piedmont sequences in italics. The nucleotide substitution model used as a prior for the MCMC was GTR + I + G according to the jModelTest2 results. The tree is mid-point routed, and the scale of branch length is expressed as substitution per site. Taxa are divided into four clusters, according to the literature: IS insect-specific; NK no-known vector; MB mosquito-borne; TB tick-borne.
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Fig3: Phylogenetic tree based on the long NS5 fragment of the Flavivirus genus. A fragment of 937 bp of the NS5 gene was used to infer the tree. The phylogenetic analysis includes 35 reference sequences from GenBank and 20 from this study. Unpublished sequences are given in bold, Piedmont sequences in italics. The nucleotide substitution model used as a prior for the MCMC was GTR + I + G according to the jModelTest2 results. The tree is mid-point routed, and the scale of branch length is expressed as substitution per site. Taxa are divided into four clusters, according to the literature: IS insect-specific; NK no-known vector; MB mosquito-borne; TB tick-borne.

Mentions: The five USUV-positive pools were composed of Cx. pipiens individuals, all collected in Novara province. In 2012, three pools were sampled on August 22nd (two of which from the same trap in the city of Novara), while in the following session (September 5th) a positive pool was collected in a different location 5 km away from the first ones. The only USUV-positive pool detected in 2011 came from Cameri in Novara province. Positivity was confirmed by USUV-specific PCR assay targeting a portion of the envelope protein (env) gene. The partial env sequences (GenBank: KF801585-88 and KF882515), almost identical to each other, resulted 99% similar to sequences retrieved from Cx. pipiens in Piedmont in 2009 (GenBank: JN257983) and 2010 (GenBank: JN257982), as well as to a sequence retrieved from blackbirds in Vienna in 2001 (GenBank: AY453411). However, the similarity decreased to 96% when compared to the SAAR 1776 strain isolated from mosquitoes in South Africa in 1958 (GenBank: AY453412). In the phylogenetic trees of the Flavivirus genus, the five USUV strains clustered together with Italian strains previously found in Piedmont and bordering regions (Figures 2 and 3).Figure 2


Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011-2012.

Rizzo F, Cerutti F, Ballardini M, Mosca A, Vitale N, Radaelli MC, Desiato R, Prearo M, Pautasso A, Casalone C, Acutis P, Peletto S, Mandola ML - Parasit Vectors (2014)

Phylogenetic tree based on the long NS5 fragment of the Flavivirus genus. A fragment of 937 bp of the NS5 gene was used to infer the tree. The phylogenetic analysis includes 35 reference sequences from GenBank and 20 from this study. Unpublished sequences are given in bold, Piedmont sequences in italics. The nucleotide substitution model used as a prior for the MCMC was GTR + I + G according to the jModelTest2 results. The tree is mid-point routed, and the scale of branch length is expressed as substitution per site. Taxa are divided into four clusters, according to the literature: IS insect-specific; NK no-known vector; MB mosquito-borne; TB tick-borne.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150984&req=5

Fig3: Phylogenetic tree based on the long NS5 fragment of the Flavivirus genus. A fragment of 937 bp of the NS5 gene was used to infer the tree. The phylogenetic analysis includes 35 reference sequences from GenBank and 20 from this study. Unpublished sequences are given in bold, Piedmont sequences in italics. The nucleotide substitution model used as a prior for the MCMC was GTR + I + G according to the jModelTest2 results. The tree is mid-point routed, and the scale of branch length is expressed as substitution per site. Taxa are divided into four clusters, according to the literature: IS insect-specific; NK no-known vector; MB mosquito-borne; TB tick-borne.
Mentions: The five USUV-positive pools were composed of Cx. pipiens individuals, all collected in Novara province. In 2012, three pools were sampled on August 22nd (two of which from the same trap in the city of Novara), while in the following session (September 5th) a positive pool was collected in a different location 5 km away from the first ones. The only USUV-positive pool detected in 2011 came from Cameri in Novara province. Positivity was confirmed by USUV-specific PCR assay targeting a portion of the envelope protein (env) gene. The partial env sequences (GenBank: KF801585-88 and KF882515), almost identical to each other, resulted 99% similar to sequences retrieved from Cx. pipiens in Piedmont in 2009 (GenBank: JN257983) and 2010 (GenBank: JN257982), as well as to a sequence retrieved from blackbirds in Vienna in 2001 (GenBank: AY453411). However, the similarity decreased to 96% when compared to the SAAR 1776 strain isolated from mosquitoes in South Africa in 1958 (GenBank: AY453412). In the phylogenetic trees of the Flavivirus genus, the five USUV strains clustered together with Italian strains previously found in Piedmont and bordering regions (Figures 2 and 3).Figure 2

Bottom Line: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment.Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown.These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

View Article: PubMed Central - PubMed

Affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, I-10154 Torino, Italy. simone.peletto@izsto.it.

ABSTRACT

Background: The genus Flavivirus comprises several mosquito-borne species, including the zoonotic pathogens West Nile and Usutu virus, circulating in animals and humans in Italy since 1998. Due to its ecological and geographical features, Piedmont is considered a risk area for flavivirus transmission. Here we report the results of a flavivirus survey (detection and genetic characterization) of mosquitoes collected in Piedmont in 2012 and the genetic characterization of three strains detected in 2011.

Methods: Pools of 1-203 mosquitoes, upon RNA extraction with TRIzol, were screened by a PCR assay for a 263 bp fragment of the Flavivirus NS5 gene. All positive samples were tested with a specific PCR for the E protein gene of Usutu virus and a generic Flavivirus RT-nested-PCR for a larger tract of the NS5 gene before sequencing. Phylogenetic trees were built with both NS5 fragments of representative Flavivirus species. DNA extracts of part of the positive pools were tested to detect sequences integrated in the host genome.

Results: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment. Among the 19 sequences correlating with the insect-specific flavivirus group, ten samples, retrieved from Aedes albopictus, clustered within Aedes flavivirus, while the other nine aggregated in a separate clade composed of strains from various mosquito species (mainly Aedes vexans) from Piedmont and the Czech Republic. Six out of these nine also presented a DNA form of the sequence. The remaining sequences belonged to the mosquito-borne group: four, all from Culex pipiens, correlated to Italian Usutu virus strains, whereas two, from Ochlerotatus caspius, were highly similar to Marisma mosquito virus (MMV).

Conclusions: Our findings confirm the circulation of Usutu virus and of the potentially zoonotic Marisma mosquito virus in Piedmont. This is the first detection of Aedes flavivirus in Piedmont. Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown. These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

Show MeSH
Related in: MedlinePlus