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Viruses are infectious agents that can only replicate inside cells. Tests available to make an early and effective diagnosis of viral diseases are mainly based on PCR, the gold-standard method, or in the detection of virus antigens or antibodies against them. The need for rapid diagnostic tests postulates aptamers as an alternative to antibodies. Aptamers are short single-stranded nucleic acid molecules that are selected through an in vitro process called SELEX. They show numerous advantages over antibodies, such as greater stability and chemical synthesis that minimizes variability between batches. However, they still have some limitations, for example, their low commercial availability, or the absence of standardized protocols for their characterization. In this work, different approaches for selecting aptamers against viruses are presented, as well as their targets, sequences, and binding affinities. Recent efforts aimed to develop aptamer-based methods for viral detection, using optical, electrochemical and piezoelectric transduction, are critically reviewed.
Elena Sánchez-Báscones; Francisco Parra; María Jesús Lobo-Castañón. Aptamers against viruses: Selection strategies and bioanalytical applications. TrAC Trends in Analytical Chemistry 2021, 143, 116349 .
AMA StyleElena Sánchez-Báscones, Francisco Parra, María Jesús Lobo-Castañón. Aptamers against viruses: Selection strategies and bioanalytical applications. TrAC Trends in Analytical Chemistry. 2021; 143 ():116349.
Chicago/Turabian StyleElena Sánchez-Báscones; Francisco Parra; María Jesús Lobo-Castañón. 2021. "Aptamers against viruses: Selection strategies and bioanalytical applications." TrAC Trends in Analytical Chemistry 143, no. : 116349.
In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.
Fábio Abade dos Santos; Carina Carvalho; Maria Peleteiro; Francisco Parra; Margarida Duarte. A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies. Viruses 2021, 13, 715 .
AMA StyleFábio Abade dos Santos, Carina Carvalho, Maria Peleteiro, Francisco Parra, Margarida Duarte. A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies. Viruses. 2021; 13 (4):715.
Chicago/Turabian StyleFábio Abade dos Santos; Carina Carvalho; Maria Peleteiro; Francisco Parra; Margarida Duarte. 2021. "A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies." Viruses 13, no. 4: 715.
Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In two of these seven animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P‐GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data shows the presence of Calicivirus compatible virions in the nucleus of hepatocytes, which has not been demonstrated before and constitutes a paradigm shift for caliciviruses’s replication cycle.
F.A. Abade dos Santos; A. Pinto; Thomas Burgoyne; K.P. Dalton; C.L. Carvalho; D.W. Ramilo; C. Carneiro; T. Carvalho; M.C. Peleteiro; F. Parra; M.D. Duarte. Spillover events of Rabbit Hemorrhagic Disease Virus 2 (recombinant GI.4P‐GI.2) from Lagomorpha to Eurasian badger. Transboundary and Emerging Diseases 2021, 1 .
AMA StyleF.A. Abade dos Santos, A. Pinto, Thomas Burgoyne, K.P. Dalton, C.L. Carvalho, D.W. Ramilo, C. Carneiro, T. Carvalho, M.C. Peleteiro, F. Parra, M.D. Duarte. Spillover events of Rabbit Hemorrhagic Disease Virus 2 (recombinant GI.4P‐GI.2) from Lagomorpha to Eurasian badger. Transboundary and Emerging Diseases. 2021; ():1.
Chicago/Turabian StyleF.A. Abade dos Santos; A. Pinto; Thomas Burgoyne; K.P. Dalton; C.L. Carvalho; D.W. Ramilo; C. Carneiro; T. Carvalho; M.C. Peleteiro; F. Parra; M.D. Duarte. 2021. "Spillover events of Rabbit Hemorrhagic Disease Virus 2 (recombinant GI.4P‐GI.2) from Lagomorpha to Eurasian badger." Transboundary and Emerging Diseases , no. : 1.
In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains’ ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.
Fábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus). Viruses 2020, 12, 1127 .
AMA StyleFábio A. Abade Dos Santos, Carina L. Carvalho, Andreia Pinto, Ranjit Rai, Madalena Monteiro, Paulo Carvalho, Paula Mendonça, Maria C. Peleteiro, Francisco Parra, Margarida D. Duarte. Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus). Viruses. 2020; 12 (10):1127.
Chicago/Turabian StyleFábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. 2020. "Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus)." Viruses 12, no. 10: 1127.
In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. Soon, a recombinant virus (MYXV-Tol or ha-MYXV) was identified, harboring a 2.8 kb insertion containing evolved duplicates of M060L, M061L, M064L, and M065L from MYXV. Since 2017, 1616 rabbits and 82 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ ha-MYXV strains. A subset (20%) of the positive samples was tested for the insert with MYXV being detected in rabbits and recombinant MYXV in hares. Recently, two wild rabbits found dead in South Portugal, showing skin oedema and pulmonary lesions tested positive for the 2.8 Kb insert. Sequencing showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs of both rabbits by electron microscopy, and isolation in RK13 cells showed virus infectivity. Despite the analysis of recombinant MYXV genomes may predict its ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, which raises serious concerns for the future of the Iberian wild leporids and emphasizes the need to continue monitoring MYXV and recombinant MYXV in both species.
Fábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus). 2020, 1 .
AMA StyleFábio A. Abade Dos Santos, Carina L. Carvalho, Andreia Pinto, Ranjit Rai, Madalena Monteiro, Paulo Carvalho, Paula Mendonça, Maria C. Peleteiro, Francisco Parra, Margarida D. Duarte. Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus). . 2020; ():1.
Chicago/Turabian StyleFábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. 2020. "Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus)." , no. : 1.
Gonzalo López‐Lorenzo; Cynthia López‐Novo; Alberto Prieto; José Manuel Díaz; Julián Gullón; José Luis Arnal; Alfredo Benito; Pablo Díaz; Rosario Panadero; Pablo Díez‐Baños; Kevin P. Dalton; Francisco Parra; Gonzalo Fernández. Molecular detection of myxoma virus in the environment of vaccinated rabbitries. Transboundary and Emerging Diseases 2020, 68, 1424 -1431.
AMA StyleGonzalo López‐Lorenzo, Cynthia López‐Novo, Alberto Prieto, José Manuel Díaz, Julián Gullón, José Luis Arnal, Alfredo Benito, Pablo Díaz, Rosario Panadero, Pablo Díez‐Baños, Kevin P. Dalton, Francisco Parra, Gonzalo Fernández. Molecular detection of myxoma virus in the environment of vaccinated rabbitries. Transboundary and Emerging Diseases. 2020; 68 (3):1424-1431.
Chicago/Turabian StyleGonzalo López‐Lorenzo; Cynthia López‐Novo; Alberto Prieto; José Manuel Díaz; Julián Gullón; José Luis Arnal; Alfredo Benito; Pablo Díaz; Rosario Panadero; Pablo Díez‐Baños; Kevin P. Dalton; Francisco Parra; Gonzalo Fernández. 2020. "Molecular detection of myxoma virus in the environment of vaccinated rabbitries." Transboundary and Emerging Diseases 68, no. 3: 1424-1431.
In this retrospective study, we describe the relative occurrence of clinical myxomatosis, and rabbit haemorrhagic disease (RHD), on 1714 commercial farms visited in Spain, between 1988 and 2018. We determined the annual prevalence based on 817 visits to 394 farms affected by myxomatosis. Myxomatosis was more prevalent from August to March, being lowest in June (3%) and highest in September (8.9%). With regard to RHD, we assessed 253 visits to 156 affected farms. We analyzed mean annual and monthly incidence. Two important RHD epidemics occurred; the first in 1988–1989 due to RHDV GI.1 (also known as RHDV), and the second from 2011 to 2013 due to RHDV GI.2 (RHDV2 or RHDVb). These epidemics occurred at times when effective vaccination had not been carried out. Relative monthly incidence in 2011–2018 was higher from April to August (p < 0.001). The results we obtained from 1404 necropsies on 102 farms did not clearly relate serosanguinous nasal discharge in rabbits with disease caused by GI.2 infection. We also assessed vaccination schedules used on 200 doe farms visited from the end of 2014 to 2018; 95.5% vaccinated against myxomatosis and 97.5% against RHD. Both diseases remain prevalent; however, effective vaccination has produced a steady decline in myxomatosis and RHDV GI.1 and GI.2 on-farm detection. The maintenance of high hygienic standards will be needed to continue and improve this control. However, further studies are required to investigate the causes of sustained virus presence and vaccine breaks.
Joan M. Rosell; L. Fernando De La Fuente; Francisco Parra; Kevin P. Dalton; J. Ignacio Badiola Sáiz; Ana Pérez De Rozas; Juan J. Badiola Díez; Daniel Fernández De Luco; Jordi Casal; Natàlia Majó; Jordina Casas; Ricard Garriga; Xosé M. Fernández Magariños. Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain. Animals 2019, 9, 780 .
AMA StyleJoan M. Rosell, L. Fernando De La Fuente, Francisco Parra, Kevin P. Dalton, J. Ignacio Badiola Sáiz, Ana Pérez De Rozas, Juan J. Badiola Díez, Daniel Fernández De Luco, Jordi Casal, Natàlia Majó, Jordina Casas, Ricard Garriga, Xosé M. Fernández Magariños. Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain. Animals. 2019; 9 (10):780.
Chicago/Turabian StyleJoan M. Rosell; L. Fernando De La Fuente; Francisco Parra; Kevin P. Dalton; J. Ignacio Badiola Sáiz; Ana Pérez De Rozas; Juan J. Badiola Díez; Daniel Fernández De Luco; Jordi Casal; Natàlia Majó; Jordina Casas; Ricard Garriga; Xosé M. Fernández Magariños. 2019. "Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain." Animals 9, no. 10: 780.
The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host–pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis‐like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5‐month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next‐generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population.
Kevin P. Dalton; José M. Martín; Inés Nicieza; Ana Podadera; Daniel De Llano; Rosa Casais; Salvador Gimenez; Ignacio Badiola; Montserrat Agüero; Manuel Duran; Dolores Buitrago; Luis J. Romero; Elena García; Francisco Parra. Myxoma virusjumps species to the Iberian hare. Transboundary and Emerging Diseases 2019, 66, 2218 -2226.
AMA StyleKevin P. Dalton, José M. Martín, Inés Nicieza, Ana Podadera, Daniel De Llano, Rosa Casais, Salvador Gimenez, Ignacio Badiola, Montserrat Agüero, Manuel Duran, Dolores Buitrago, Luis J. Romero, Elena García, Francisco Parra. Myxoma virusjumps species to the Iberian hare. Transboundary and Emerging Diseases. 2019; 66 (6):2218-2226.
Chicago/Turabian StyleKevin P. Dalton; José M. Martín; Inés Nicieza; Ana Podadera; Daniel De Llano; Rosa Casais; Salvador Gimenez; Ignacio Badiola; Montserrat Agüero; Manuel Duran; Dolores Buitrago; Luis J. Romero; Elena García; Francisco Parra. 2019. "Myxoma virusjumps species to the Iberian hare." Transboundary and Emerging Diseases 66, no. 6: 2218-2226.
RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease.
K.P. Dalton; A. Balseiro; R.A. Juste; A. Podadera; I. Nicieza; D. del Llano; R. González; J.M. Martin Alonso; J.M. Prieto; F. Parra; R. Casais. Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread. Veterinary Microbiology 2018, 220, 24 -32.
AMA StyleK.P. Dalton, A. Balseiro, R.A. Juste, A. Podadera, I. Nicieza, D. del Llano, R. González, J.M. Martin Alonso, J.M. Prieto, F. Parra, R. Casais. Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread. Veterinary Microbiology. 2018; 220 ():24-32.
Chicago/Turabian StyleK.P. Dalton; A. Balseiro; R.A. Juste; A. Podadera; I. Nicieza; D. del Llano; R. González; J.M. Martin Alonso; J.M. Prieto; F. Parra; R. Casais. 2018. "Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread." Veterinary Microbiology 220, no. : 24-32.
Since its emergence, variant RHDV (RHDVb/RHDV2) has spread throughout the Iberian Peninsula aided by the apparent lack of cross protection provided by classic (genogroup 1; G1) strain derived vaccines. In addition to RHDVb, full-length genome sequencing of RHDV strains has recently revealed the circulation of recombinant viruses on the Iberian Peninsula. These recombinant viruses contain the RHDVb structural protein encoding sequences and the non-structural coding regions of either pathogenic RHDV-G1 strains or non-pathogenic (np) rabbit caliciviruses. The aim of the work was twofold: firstly to validate a diagnostic real time RT-PCR developed in 2012 for the detection of RHDVb strains and secondly, to design a conventional RT-PCR for the differentiation of RHDVb strains from RHDVb recombinants by subsequent sequencing of the amplicon.
K.P. Dalton; J.L. Arnal; A.A. Benito; G. Chacón; J.M. Martín Alonso; F. Parra. Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants. Journal of Virological Methods 2018, 251, 118 -122.
AMA StyleK.P. Dalton, J.L. Arnal, A.A. Benito, G. Chacón, J.M. Martín Alonso, F. Parra. Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants. Journal of Virological Methods. 2018; 251 ():118-122.
Chicago/Turabian StyleK.P. Dalton; J.L. Arnal; A.A. Benito; G. Chacón; J.M. Martín Alonso; F. Parra. 2018. "Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants." Journal of Virological Methods 251, no. : 118-122.
Myxomatosis is a viral disease that affects European rabbits (Oryctolagus cuniculus) worldwide. In Spain, populations of wild rabbits drastically decreased in the 1950s after the first outbreak of myxomatosis. Since that first appearance, it seems to be an annual epizootic in Spain with periodic outbreaks, predominantly in summer and autumn. Taking into account rabbit population structure, abundance, and genetic lineage, this paper attempts to make a large-scale characterization of myxomatosis seroprevalence based on the immune status of 29 rabbit populations distributed throughout Spain, where O. cuniculus cuniculus and O. c. algirus, the two known rabbit subspecies, naturally inhabit. A total of 654 samples were collected between 2003 and 2009, and seroprevalence of antibodies against Myxoma virus (MYXV) was determined. Overall, our results revealed that 53% of the rabbit samples were positive to antibodies against MYXV. Newborn and juvenile rabbits were the most susceptible animals to the virus, with 19% and 16% seropositivity for newborn and juveniles, respectively, while adult rabbits were the most protected, with 65% of seropositive samples. This suggests that prevalence is negatively related to the proportion of newborn and juvenile rabbits in a population. Our results also showed that seroprevalence against MYXV tended to be higher in high-abundance populations. In contrast, no differences were detected in seroprevalence between rabbit subspecies. This study confirms that >60years since first outbreak, myxomatosis is an endemic disease in Spain. Based on the results, the establishment of a myxomatosis surveillance protocol is proposed.
Rafael Villafuerte; Francisca Castro; Esther Ramírez; Irene Cotilla; Francisco Parra; Miguel Delibes-Mateos; Pilar Recuerda; Carlos Rouco. Large-scale assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60 years after first outbreak in Spain. Research in Veterinary Science 2017, 114, 281 -286.
AMA StyleRafael Villafuerte, Francisca Castro, Esther Ramírez, Irene Cotilla, Francisco Parra, Miguel Delibes-Mateos, Pilar Recuerda, Carlos Rouco. Large-scale assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60 years after first outbreak in Spain. Research in Veterinary Science. 2017; 114 ():281-286.
Chicago/Turabian StyleRafael Villafuerte; Francisca Castro; Esther Ramírez; Irene Cotilla; Francisco Parra; Miguel Delibes-Mateos; Pilar Recuerda; Carlos Rouco. 2017. "Large-scale assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60 years after first outbreak in Spain." Research in Veterinary Science 114, no. : 281-286.
The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10 ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4 × 108molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.
K.P. Dalton; A. Podadera; V. Granda; I. Nicieza; D. Del Llano; R. González; J.R. De Los Toyos; M. García Ocaña; F. Vázquez; J.M. Martín Alonso; J.M. Prieto; F. Parra; R. Casais. ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts. Journal of Virological Methods 2017, 251, 38 -42.
AMA StyleK.P. Dalton, A. Podadera, V. Granda, I. Nicieza, D. Del Llano, R. González, J.R. De Los Toyos, M. García Ocaña, F. Vázquez, J.M. Martín Alonso, J.M. Prieto, F. Parra, R. Casais. ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts. Journal of Virological Methods. 2017; 251 ():38-42.
Chicago/Turabian StyleK.P. Dalton; A. Podadera; V. Granda; I. Nicieza; D. Del Llano; R. González; J.R. De Los Toyos; M. García Ocaña; F. Vázquez; J.M. Martín Alonso; J.M. Prieto; F. Parra; R. Casais. 2017. "ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts." Journal of Virological Methods 251, no. : 38-42.
Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be critical for the infection process. Therefore, we have determined binding epitopes of synthetic type 1 to 6 blood group A- and B-tetrasaccharides binding to GII.4 human Norovirus virus like particles (VLPs) using STD NMR experiments. So far, little information is available from crystal structure analysis studies on the interactions of the reducing-end sugars with the protruding domain (P-domain) of the viral coat protein VP1. Here, we show that the reducing-end sugars make notable contacts with the protein surface. The type of glycosidic linkage, and the identity of the sugar at the reducing end modulate HBGA recognition. Most strikingly, type 2 structures yield only very poor saturation transfer indicating impeded binding. This observation is in accordance with previous mass spectrometry based affinity measurements, and can be understood based on recent crystal structure data of a complex of highly homologous GII.4 P-dimers with H-type 2 trisaccharide where the N-acetyl group of the reducing N-acetyl glucosamine residue points towards a loop comprising amino acids Q390 to H395. We suggest that in our case, binding of type 2 A- and B-tetrasaccharides leads to steric conflicts with this loop. In order to identify factors determining L-Fuc recognition, we also synthesized GII.4 VLPs with point mutations D391A and H395A. Prior studies had suggested that these residues, located in a second shell around the L-Fuc binding site, assist L-Fuc binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes suggesting that these two mutations do not significantly alter HBGA recognition. Our study emphasizes that recognition of α−(1→2)-linked L-Fuc residues is a conserved feature of GII.4 noroviruses. However, structural variation of the HBGA core structures clearly modulates molecular recognition depending on the genotype.
Brigitte Fiege; Mila Leuthold; Francisco Parra; Kevin P. Dalton; Peter J. Meloncelli; Todd L. Lowary; Thomas Peters. Epitope mapping of histo blood group antigens bound to norovirus VLPs using STD NMR experiments reveals fine details of molecular recognition. Glycoconjugate Journal 2017, 34, 679 -689.
AMA StyleBrigitte Fiege, Mila Leuthold, Francisco Parra, Kevin P. Dalton, Peter J. Meloncelli, Todd L. Lowary, Thomas Peters. Epitope mapping of histo blood group antigens bound to norovirus VLPs using STD NMR experiments reveals fine details of molecular recognition. Glycoconjugate Journal. 2017; 34 (5):679-689.
Chicago/Turabian StyleBrigitte Fiege; Mila Leuthold; Francisco Parra; Kevin P. Dalton; Peter J. Meloncelli; Todd L. Lowary; Thomas Peters. 2017. "Epitope mapping of histo blood group antigens bound to norovirus VLPs using STD NMR experiments reveals fine details of molecular recognition." Glycoconjugate Journal 34, no. 5: 679-689.
Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970–1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.
Jacques Le Pendu; Joana Abrantes; Stéphane Bertagnoli; Jean-Sébastien Guitton; Ghislaine Le Gall-Reculé; Ana Lopes; Stéphane Marchandeau; Fernando Alda; Tereza Almeida; Alves Paulo Célio; Juan Barcena; Galina Burmakina; Esther Blanco; Carlos Calvete; Patrizia Cavadini; Brian Cooke; Kevin P. Dalton; Miguel Delibes Mateos; Wiesław Deptuła; John-Sebastian Eden; Fang Wang; Catarina C Ferreira; Paula G Ferreira; Pilar Foronda; Paulo Célio Alves; Dolores Gavier-Widén; Robyn Hall; Beata Hukowska-Szematowicz; Peter Kerr; John Kovaliski; Antonio Lavazza; Jackie Mahar; Alexander Malogolovkin; Raquel M. Marques; Sara Marques; Aaron Martin-Alonso; Pedro Monterroso; Sacramento Moreno; Greg Mutze; Aleksija Neimanis; Paulina Niedźwiedzka-Rystwej; David Peacock; Francisco Parra; Mara Rocchi; Carlos Rouco; Nathalie Ruvoën-Clouet; Eliane Silva; Diogo Silvério; Tanja Strive; Gertrude Thompson; Beata Tokarz-Deptuła; Pedro Esteves. Proposal for a unified classification system and nomenclature of lagoviruses. Journal of General Virology 2017, 98, 1658 -1666.
AMA StyleJacques Le Pendu, Joana Abrantes, Stéphane Bertagnoli, Jean-Sébastien Guitton, Ghislaine Le Gall-Reculé, Ana Lopes, Stéphane Marchandeau, Fernando Alda, Tereza Almeida, Alves Paulo Célio, Juan Barcena, Galina Burmakina, Esther Blanco, Carlos Calvete, Patrizia Cavadini, Brian Cooke, Kevin P. Dalton, Miguel Delibes Mateos, Wiesław Deptuła, John-Sebastian Eden, Fang Wang, Catarina C Ferreira, Paula G Ferreira, Pilar Foronda, Paulo Célio Alves, Dolores Gavier-Widén, Robyn Hall, Beata Hukowska-Szematowicz, Peter Kerr, John Kovaliski, Antonio Lavazza, Jackie Mahar, Alexander Malogolovkin, Raquel M. Marques, Sara Marques, Aaron Martin-Alonso, Pedro Monterroso, Sacramento Moreno, Greg Mutze, Aleksija Neimanis, Paulina Niedźwiedzka-Rystwej, David Peacock, Francisco Parra, Mara Rocchi, Carlos Rouco, Nathalie Ruvoën-Clouet, Eliane Silva, Diogo Silvério, Tanja Strive, Gertrude Thompson, Beata Tokarz-Deptuła, Pedro Esteves. Proposal for a unified classification system and nomenclature of lagoviruses. Journal of General Virology. 2017; 98 (7):1658-1666.
Chicago/Turabian StyleJacques Le Pendu; Joana Abrantes; Stéphane Bertagnoli; Jean-Sébastien Guitton; Ghislaine Le Gall-Reculé; Ana Lopes; Stéphane Marchandeau; Fernando Alda; Tereza Almeida; Alves Paulo Célio; Juan Barcena; Galina Burmakina; Esther Blanco; Carlos Calvete; Patrizia Cavadini; Brian Cooke; Kevin P. Dalton; Miguel Delibes Mateos; Wiesław Deptuła; John-Sebastian Eden; Fang Wang; Catarina C Ferreira; Paula G Ferreira; Pilar Foronda; Paulo Célio Alves; Dolores Gavier-Widén; Robyn Hall; Beata Hukowska-Szematowicz; Peter Kerr; John Kovaliski; Antonio Lavazza; Jackie Mahar; Alexander Malogolovkin; Raquel M. Marques; Sara Marques; Aaron Martin-Alonso; Pedro Monterroso; Sacramento Moreno; Greg Mutze; Aleksija Neimanis; Paulina Niedźwiedzka-Rystwej; David Peacock; Francisco Parra; Mara Rocchi; Carlos Rouco; Nathalie Ruvoën-Clouet; Eliane Silva; Diogo Silvério; Tanja Strive; Gertrude Thompson; Beata Tokarz-Deptuła; Pedro Esteves. 2017. "Proposal for a unified classification system and nomenclature of lagoviruses." Journal of General Virology 98, no. 7: 1658-1666.
Virus entry depends on biomolecular recognition at the surface of cell membranes. In the case of glycolipid receptors, these events are expected to be influenced by how the glycan epitope close to the membrane is presented to the virus. This presentation of membrane-associated glycans is more restricted than that of glycans in solution, particularly because of orientational constraints imposed on the glycolipid through its lateral interactions with other membrane lipids and proteins. We have developed and employed a total internal reflection fluorescence microscopy-based binding assay and a scheme for molecular dynamics (MD) membrane simulations to investigate the consequences of various glycan presentation effects. The system studied was histo-blood group antigen (HBGA) epitopes of membrane-bound glycosphingolipids (GSLs) derived from small intestinal epithelium of humans (type 1 chain) and dogs (type 2 chain) interacting with GII.4 norovirus-like particles. Our experimental results showed strong binding to all lipid-linked type 1 chain HBGAs but no or only weak binding to the corresponding type 2 chain HBGAs. This is in contrast to results derived from STD experiments with free HBGAs in solution where binding was observed for Lewis x. The MD data suggest that the strong binding to type 1 chain glycolipids was due to the well-exposed (1,2)-linked α-l-Fucp and (1,4)-linked α-l-Fucp residues, while the weaker binding or lack of binding to type 2 chain HBGAs was due to the very restricted accessibility of the (1,3)-linked α-l-Fucp residue when the glycolipid is embedded in a phospholipid membrane. Our results not only contribute to a general understanding of protein–carbohydrate interactions on model membrane surfaces, particularly in the context of virus binding, but also suggest a possible role of human intestinal GSLs as potential receptors for norovirus uptake.
Waqas Nasir; Martin Frank; Angelika Kunze; Marta Bally; Francisco Parra; Per-Georg Nyholm; Fredrik Höök; Göran Larson. Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes. ACS Chemical Biology 2017, 12, 1288 -1296.
AMA StyleWaqas Nasir, Martin Frank, Angelika Kunze, Marta Bally, Francisco Parra, Per-Georg Nyholm, Fredrik Höök, Göran Larson. Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes. ACS Chemical Biology. 2017; 12 (5):1288-1296.
Chicago/Turabian StyleWaqas Nasir; Martin Frank; Angelika Kunze; Marta Bally; Francisco Parra; Per-Georg Nyholm; Fredrik Höök; Göran Larson. 2017. "Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes." ACS Chemical Biology 12, no. 5: 1288-1296.
This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross-reactivity with other RHDV types giving a specific result in under 10 min using rabbit liquid exudates or liver homogenate samples taken at necropsy.
K. P. Dalton; I. Nicieza; A. Podadera; D. Llano; J. M. Martin Alonso; Juan De Los Toyos; M. García Ocaña; F. Vázquez‐Villa; B. Velasco; O. Landeta; F. Parra. Fast specific field detection of RHDVb. Transboundary and Emerging Diseases 2017, 65, 232 -234.
AMA StyleK. P. Dalton, I. Nicieza, A. Podadera, D. Llano, J. M. Martin Alonso, Juan De Los Toyos, M. García Ocaña, F. Vázquez‐Villa, B. Velasco, O. Landeta, F. Parra. Fast specific field detection of RHDVb. Transboundary and Emerging Diseases. 2017; 65 (1):232-234.
Chicago/Turabian StyleK. P. Dalton; I. Nicieza; A. Podadera; D. Llano; J. M. Martin Alonso; Juan De Los Toyos; M. García Ocaña; F. Vázquez‐Villa; B. Velasco; O. Landeta; F. Parra. 2017. "Fast specific field detection of RHDVb." Transboundary and Emerging Diseases 65, no. 1: 232-234.
Little is known about the antiviral response in mollusks. As in other invertebrates, the interferon signaling pathways have not been identified, and in fact, there is a debate about whether invertebrates possess antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naive mussels confer resistance in oysters to ostreid herpesvirus 1 (OsHV-1) when oyster hemocytes are incubated with mussel hemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel hemolymph and hemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel hemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible for the antiviral activity of mussel hemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks. Moreover, myticin C peptides showed antiviral activity against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications. IMPORTANCE Several bioactive molecules that have potential pharmaceutical or industrial applications have been identified and isolated from marine invertebrates. Myticin C, an antimicrobial peptide from the Mediterranean mussel ( Mytilus galloprovincialis ) that was identified by proteomic techniques in both mussel hemolymph and hemocytes, showed potential as an antiviral agent against ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster-farming sector. Both hemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster hemocytes. Additionally, a modified peptide derived from myticin C or the nanoencapsulated normal peptide also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of mollusks.
Beatriz Novoa; Alejandro Romero; Ángel L. Álvarez; Rebeca Moreira; Patricia Pereiro; María M. Costa; Sonia Dios; Amparo Estepa; Francisco Parra; Antonio Figueras. Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses. Journal of Virology 2016, 90, 7692 -7702.
AMA StyleBeatriz Novoa, Alejandro Romero, Ángel L. Álvarez, Rebeca Moreira, Patricia Pereiro, María M. Costa, Sonia Dios, Amparo Estepa, Francisco Parra, Antonio Figueras. Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses. Journal of Virology. 2016; 90 (17):7692-7702.
Chicago/Turabian StyleBeatriz Novoa; Alejandro Romero; Ángel L. Álvarez; Rebeca Moreira; Patricia Pereiro; María M. Costa; Sonia Dios; Amparo Estepa; Francisco Parra; Antonio Figueras. 2016. "Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses." Journal of Virology 90, no. 17: 7692-7702.
Recently, combined nuclear magnetic resonance (NMR), native MS and X-ray crystallographic studies have demonstrated that binding of histo-blood group antigens (HBGAs) to norovirus capsid protein (P-dimers) is a cooperative process involving four binding pockets. Here, we show that binding to norovirus virus-like particles (VLPs) is even more complex. We performed sexually transmitted disease NMR titration experiments with two representative genotypes of norovirus VLPs using l-fucose as a minimal HBGA. Compared to titrations with P-dimers, the corresponding binding isotherms reflect at least six distinct binding events.
Alvaro Mallagaray; Christoph Rademacher; Francisco Parra; Grant Hansman; Thomas Peters. Saturation transfer difference nuclear magnetic resonance titrations reveal complex multistep-binding of l-fucose to norovirus particles. Glycobiology 2016, 27, 80 -86.
AMA StyleAlvaro Mallagaray, Christoph Rademacher, Francisco Parra, Grant Hansman, Thomas Peters. Saturation transfer difference nuclear magnetic resonance titrations reveal complex multistep-binding of l-fucose to norovirus particles. Glycobiology. 2016; 27 (1):80-86.
Chicago/Turabian StyleAlvaro Mallagaray; Christoph Rademacher; Francisco Parra; Grant Hansman; Thomas Peters. 2016. "Saturation transfer difference nuclear magnetic resonance titrations reveal complex multistep-binding of l-fucose to norovirus particles." Glycobiology 27, no. 1: 80-86.
Despite the success of vaccination against myxoma virus, myxomatosis remains a problem on rabbit farms throughout Spain and Europe. In this study we set out to evaluate possible causes of myxoma virus (MYXV) vaccine failures addressing key issues with regard to pathogen, vaccine and vaccination strategies. This was done by genetically characterising MYXV field isolates from farm outbreaks, selecting a representative strain for which to assay its virulence and measuring the protective capability of a commercial vaccine against this strain. Finally, we compare methods (route) of vaccine administration under farm conditions and evaluate immune response in vaccinated rabbits. The data presented here show that the vaccine tested is capable of eliciting protection in rabbits that show high levels of seroconversion. However, the number of animals failing to seroconvert following subcutaneous vaccination may leave a large number of rabbits unprotected following vaccine administration. Successful vaccination requires the strict implication of workable, planned, on farm programs. Following this, analysis to confirm seroconversion rates may be advisable. Factors such as the wild rabbit reservoir, control of biting insects and good hygienic practices must be taken into consideration to prevent vaccine failures from occurring.
K.P. Dalton; I. Nicieza; D. De Llano; J. Gullón; M. Inza; M. Petralanda; Z. Arroita; Francisco Parra. Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms. Veterinary Microbiology 2015, 178, 208 -216.
AMA StyleK.P. Dalton, I. Nicieza, D. De Llano, J. Gullón, M. Inza, M. Petralanda, Z. Arroita, Francisco Parra. Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms. Veterinary Microbiology. 2015; 178 (3-4):208-216.
Chicago/Turabian StyleK.P. Dalton; I. Nicieza; D. De Llano; J. Gullón; M. Inza; M. Petralanda; Z. Arroita; Francisco Parra. 2015. "Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms." Veterinary Microbiology 178, no. 3-4: 208-216.
Rabbit hemorrhagic disease virus (RHDV), a Lagovirus of the family Caliciviridae, causes rabbit hemorrhagic disease (RHD) in the European rabbit (Oryctolagus cuniculus). The disease was first documented in 1984 in China and rapidly spread worldwide. In 2010, a new RHDV variant emerged, tentatively classified as 'RHDVb'. RHDVb is characterized by affecting vaccinated rabbits and those <2 months old, and is genetically distinct (~20 %) from older strains. To determine the evolution of RHDV, including the new variant, we generated 28 full-genome sequences from samples collected between 1994 and 2014. Phylogenetic analysis of the gene encoding the major capsid protein, VP60, indicated that all viruses sampled from 2012 to 2014 were RHDVb. Multiple recombination events were detected in the more recent RHDVb genomes, with a single major breakpoint located in the 5' region of VP60. This breakpoint divides the genome into two regions: one that encodes the non-structural proteins and another that encodes the major and minor structural proteins, VP60 and VP10, respectively. Additional phylogenetic analysis of each region revealed two types of recombinants with distinct genomic backgrounds. Recombinants always include the structural proteins of RHDVb, with non-structural proteins from non-pathogenic lagoviruses or from pathogenic genogroup 1 strains. Our results show that in contrast to the evolutionary history of older RHDV strains, recombination plays an important role in generating diversity in the newly emerged RHDVb.
Ana Lopes; Kevin P. Dalton; Maria J. Magalhães; Francisco Parra; Pedro Esteves; Edward Holmes; Joana Abrantes. Full genomic analysis of new variant rabbit hemorrhagic disease virus revealed multiple recombination events. Journal of General Virology 2015, 96, 1309 -1319.
AMA StyleAna Lopes, Kevin P. Dalton, Maria J. Magalhães, Francisco Parra, Pedro Esteves, Edward Holmes, Joana Abrantes. Full genomic analysis of new variant rabbit hemorrhagic disease virus revealed multiple recombination events. Journal of General Virology. 2015; 96 (6):1309-1319.
Chicago/Turabian StyleAna Lopes; Kevin P. Dalton; Maria J. Magalhães; Francisco Parra; Pedro Esteves; Edward Holmes; Joana Abrantes. 2015. "Full genomic analysis of new variant rabbit hemorrhagic disease virus revealed multiple recombination events." Journal of General Virology 96, no. 6: 1309-1319.