Papers by Guillermo Risatti
Virology, Nov 1, 2006
Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residu... more Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). In vitro growth of mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) was similar to parental BICv, while mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence.
Viruses, Dec 13, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
International Journal of Infectious Diseases, Dec 1, 2016
Purpose: Flaviviruses like West Nile, Zika, yellow fever and dengue virus produce an abundant non... more Purpose: Flaviviruses like West Nile, Zika, yellow fever and dengue virus produce an abundant non-coding subgenomic flavivirus RNA (sfRNA) in infected cells. SfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3'UTR of the viral genomic RNA. SfRNA production is conserved in insect-specific, mosquito-and tick-borne, as well as no-known-vector flaviviruses, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here we investigated the function of sfRNA during West Nile virus (WNV) infection of Culex pipiens mosquitoes and evaluated its role in determining virus transmission. Methods & Materials: An sfRNA-deficient WNV was generated and compared to wildtype WNV for replication rates in cell lines. Infectivity and transmissibility was assessed in mosquitoes by measuring virus titers in the body and saliva. Results: The sfRNA-deficient WNV displayed similar growth kinetics as wildtype WNV in both RNAi-competent and RNAicompromised mosquito cell lines. Importantly, we demonstrate that the sfRNA-deficient virus displayed significantly decreased mosquito infection and transmission rates in vivo when administrated via the blood meal. Infection and transmission rates were not affected by sfRNA after intrathoracic injection. Conclusion: This study identified sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of viral, non-coding RNA in mosquitoes, providing an explanation for the strict conservation of sfRNA production in all flaviviruses.
Mitochondrial DNA Part B, Jul 19, 2021
Abstract Haemaphysalis longicornis (Ixodida: Ixodidae), the Asian longhorned tick, which is nativ... more Abstract Haemaphysalis longicornis (Ixodida: Ixodidae), the Asian longhorned tick, which is native to temperate East Asia, has been recently detected in the northeastern region of the United States, drawing concerns about its potential impact on the US animal and public health sectors. Knowledge about the genetic features of H. longicornis found in the US is limited. Therefore, we sequenced the complete mitochondrial genome (mt-genome) from two H. longicornis ticks recently collected in the State of New York, USA, in 2020. These ticks were morphologically identified and tested for tick-borne pathogens at the Connecticut Veterinary Medical Diagnostic Laboratory (Storrs, CT). The mt-genome was 14,694 bp in length and encoded 37 genes, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogenetic analysis showed that the mt-genome clustered with those of other H. longicornis identified in China. The mt-genome sequence was 99.7% identical to a H. longicornis mt-genome (GenBank: MK439888) collected in China. The cox1 gene haplotype in these ticks belonged to the H1 type, which is the dominant haplotype present in central NJ and Staten Island, NY. The complete mt-genome data are needed to provide insights into genetic changes and phylogenetic studies of H. longicornis ticks.
Viruses, Oct 23, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Frontiers in Veterinary Science
Antibiotics, 2021
Salmonella enterica subsp. houtenae (S. houtenae) is a common subspecies in reptiles and has been... more Salmonella enterica subsp. houtenae (S. houtenae) is a common subspecies in reptiles and has been implicated as a source of serious and life-threatening diseases in humans. Although occurrence and significance of S. houtenae infections have been extensively studied, the genetic features of S. houtenae have remained unknown due to a lack of available high-quality genome sequences. We obtained the complete genome sequence of S. houtenae 45:g,z51:- strain 20-369 isolated from multiple abdominal abscesses of an African fat-tailed gecko (Hemitheconyx caudicinctus) using Nanopore and Illumina sequencing technologies and generated the 4.65Mbp complete genome sequence of the S. houtenae str. 20-369. We annotated and analyzed the genome sequence with the aim to gain a deeper understanding of the genome characteristics associated with its pathogenicity. Overall, this study found several interesting genomic features such as pseudogene formation, virulence gene profile, and novel genomic island...
Revue scientifique et technique, 2020
KEEN The factors associated with the spread and persistence of African swine fever (ASF) in the C... more KEEN The factors associated with the spread and persistence of African swine fever (ASF) in the Caucasus region remain to be fully identified. It is assumed that large naive populations of domestic free-ranging and wild pigs are critical to disease transmission and maintenance. Nonetheless, 11 years since its epidemic introduction into the region in 2007, ASF virus (ASFV) is still circulating, suggesting that an endemic cycle has been established based on contact between free-ranging domestic pigs and wild pigs, and that native Ornithodoros ticks probably serve as reservoirs for the virus. Therefore, research is required to gather information on the epidemiological status of ASF in the Caucasus region, focusing particularly on understanding modes of ASFV spread and persistence in this new virus environment. The authors established an ASFV survey targeting domestic pigs in the Tavush province of northern Armenia, an area of the country considered to be at high risk of disease incursi...
Research in Veterinary Science, 2021
Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) (sheep associated S. diarizona... more Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) (sheep associated S. diarizonae, SASd) is the most common Salmonella serotype identified in sheep flocks. Despite the involvement with animal and human infections, there is limited information regarding virulence profiles of SASds and their antibiotic resistance gene complement, particularly for those circulating in the U.S. In this study, we genetically characterized three SASds, 20-265, 20-269, and 20-312, isolated from sheep placental tissues during an abortion storm affecting a flock in Connecticut during 2020. SASds were the only bacteria isolated from analyzed sheep tissues. The isolates were sensitive to all the antibiotics tested, but all these SASd isolates carry the aminoglycoside resistance gene, aac(6')-Iaa, and a chromosomal substitution in the parC gene. The proportion of pseudogenes (5.3-5.5%) was similar among the isolates, and these SASds carry IncX1 type plasmids. Comparing with the SASds isolates from Enterobase, the three isolates showed an identical genomic virulence profile carrying virulence genes in the conserved set of other SASd isolates except for steC, iagB, iacP, sseI, and slrP genes. In the SNP-based phylogenetic analysis, SASd sequences were grouped into group A-C, and the group C was further subdivided into subgroup C1-C6. The three isolates clustered with other SASd isolates from the U.S. and Canada in subgroup C6. SASd isolates in the identical phylogenetic groups tended to have similar geographical origin. The results of our study did not provide conclusive evidence about which are the genetic traits that trigger SASds to become virulent in sheep, but our data will provide a point for comparative studies of this Salmonella serovar.
Scientific Reports, 2020
The CD2-like African swine fever virus (ASFV) gene 8DR, (also known as EP402R) encodes for a stru... more The CD2-like African swine fever virus (ASFV) gene 8DR, (also known as EP402R) encodes for a structural transmembrane glycoprotein that has been shown to mediate hemadsorption and be involved in host immunomodulation as well as the induction of protective immune response. In addition, several natural ASFV isolates showing decreased virulence in swine has been shown to be non-hemadsorbing suggesting an association between altered or deleted forms of 8DR and virus attenuation. Here we demonstrate that deletion of 8DR gene from the genome of ASFV Georgia2010 isolate (ASFV-G-Δ8DR) does not significantly alter the virulence of the virus. ASFV-G-Δ8DR inoculated intramuscularly or intranasally (in a range of 102 to 104 TCID50) produced a clinical disease in domestic pigs indistinguishable from that induced by the same doses of the virulent parental ASFV Georgia2010 isolate. In addition, viremia values in ASFV-G-Δ8DR do not differ from those detected in animals infected with parental virus....
Virology, 2019
Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical vi... more Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93-168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136-156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells.
Journal of Virology, 2016
E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several... more E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond ( 869 CKWGGNWTCV 878 , named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872 GG 873 dipeptide, as well as two aromatic residues ( 871 W and 875 W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid subst...
Virus research, Jan 4, 2015
African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral ... more African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georg...
Journal of Fish Diseases, 2012
Revue Scientifique et Technique de l'OIE, 2017
Since its introduction to the Republic of Georgia in 2007, African swine fever virus (ASFV) has s... more Since its introduction to the Republic of Georgia in 2007, African swine fever virus (ASFV) has spread across the Caucasus region, the Russian Federation, and some Eastern European countries. It is assumed that large populations of naïve, domestic, free-ranging and wild pigs are vital to the transmission of the disease. Since its epidemic emergence in the region in 2007, ASFV has continued to circulate, which suggests that an endemic cycle has been established
Viruses, 2018
We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural... more We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CA...
Virus research, Apr 29, 2018
African swine fever virus (ASFV) causes a contagious and frequently lethal disease of pigs causin... more African swine fever virus (ASFV) causes a contagious and frequently lethal disease of pigs causing significant economic consequences to the swine industry. The ASFV genome encodes for more than 150 genes, but only a few of them have been studied in detail. Here we report the characterization of open reading frame L83L which encodes a highly conserved protein across all ASFV isolates. A recombinant ASFV harboring a HA tagged L83L protein was developed (ASFV-G-L83L-HA) and used to demonstrate that L83L is a transiently expressed early virus protein. A recombinant ASFV lacking the L83L gene (ASFV-G-ΔL83L) was developed from the highly virulent field isolate Georgia2007 (ASFV-G) and was used to show that L83L is a non-essential gene. ASFV-G-ΔL83L had similar replication in primary swine macrophage cells when compared to its parental virus ASFV-G. Analysis of host-protein interactions for L83L identified IL-1β as its host ligand. Experimental infection of domestic pigs showed that ASFV-G...
PloS one, 2017
Prophylactic vaccination using live attenuated classical swine fever (CSF) vaccines has been a ve... more Prophylactic vaccination using live attenuated classical swine fever (CSF) vaccines has been a very effective method to control the disease in endemic regions and during outbreaks in previously disease-free areas. These vaccines confer effective protection against the disease at early times post-vaccination although the mechanisms mediating the protection are poorly characterized. Here we present the events occurring after the administration of our in-house developed live attenuated marker vaccine, FlagT4Gv. We previously reported that FlagT4Gv intramuscular (IM) administered conferred effective protection against intranasal challenge with virulent CSFV (BICv) as early as 7 days post-vaccination. Here we report that FlagT4Gv is able to induce protection against disease as early as three days post-vaccination. Immunohistochemical testing of tissues from FlagT4Gv-inoculated animals showed that tonsils were colonized by the vaccine virus by day 3 post-inoculation. There was a complete ...
Journal of virology, 2017
African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral ... more African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another...
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Papers by Guillermo Risatti