Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic d... more Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection.
Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronch... more Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is considered one of the most economically important viral diseases of chickens. Control of IBV has been attempted using live attenuated and inactivated vaccines. Live attenuated vaccines of the Massachusetts (Mass.) serotype are the most commonly used for this purpose. Due to the continuous emergence of new variants of the infectious bronchitis virus, the identification of the type of IBV causing an outbreak in commercial poultry is important in the selection of the appropriate vaccine(s) capable of inducing a protective immune response. The present work was aimed at developing and evaluating a duplex SYBR Green I-based real-time RT-PCR (rRT-PCR) assay for the simultaneous detection and differentiation of Mass. and non-Mass. serotypes of IBV. The duplex rRT-PCR yielded curves of amplification with two specific melting curves (Tm1 ¼ 83 C AE 0.5 C and Tm2 ¼ 87 C AE 0.5 C) and only one specific melting peak (Tm ¼ 87 C AE 0.5 C) when the IBV Mass. serotype and IBV non-Mass. serotype strains were evaluated, respectively. The detection limit of the assay was 8.2 gene copies/mL based on in vitro transcribed RNA and 0.1 EID 50 /mL. The assay was able to detect all the IBV strains assessed and discriminated well among the IBV Mass. and the IBV non-Mass. serotypes strains. In addition, amplification curves were not obtained with any of the other viruses tested. From the 300 field samples tested, the duplex rRT-PCR yielded a total of 80 samples that were positive for IBV (26.67%), 73 samples identified as the IBV Mass. serotype and seven samples as identified as the IBV non-Mass. serotype. A comparison of the performance of test as assessed with field samples revealed that the duplex rRT-PCR detected a higher number of IBV-positive samples than when conventional RT-PCR or virus isolation tests were used. The duplex rRT-PCR presented here is a useful tool for the rapid identification of outbreaks and for surveillance programmes during IB-suspected cases, particularly in countries with a vaccination control programme.
Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of... more Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of the most important diseases in swine. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. Viral miRNAs have recently been described and the number of viral miRNAs has been increasing in the past few years. In this study, small RNA libraries were constructed from two tissues of subclinically PCV2 infected pigs to explore if PCV2 can encode viral miRNAs. The deep sequencing data revealed that PCV2 does not express miRNAs in an in vivo subclinical infection.
Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adul... more Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.
In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated fr... more In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated from a reassortment event between the H1N1 pandemic influenza virus (H1N1p/2009) and endemic swine influenza virus in Cuban swine population. In November 2010, a clinical respiratory outbreak was reported on a pig fattening farm in Cuba. Phylogenetic analysis showed that all the genes of one of the isolate obtained, with the exception of neuraminidase, belonged to the H1N1p/2009 cluster. This finding suggests that H1N1pdm has been established in swine and has become a reservoir of reassortment that may produce new viruses with both animal and public health risks.
Background: Infectious bursal disease is a highly contagious and acute viral disease caused by th... more Background: Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains.
Schmallenberg virus was first detected in Germany in October 2011, associated with congenital mal... more Schmallenberg virus was first detected in Germany in October 2011, associated with congenital malformations in cattle, sheep and goats. This novel emergent agent causes mild disease in cattle with decreased milk production, fever and diarrhea. In March 2012, the German Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, reported the development and validation of a real-time RT-PCR for the diagnosis of this new virus. The Animal Virology Laboratory at the National Center for Animal and Plant Health in Cuba has adapted the protocol previously reported on the LightCycler platforms using two different mix conditions. In all cases, amplification curves obtained were specific and all the dilutions tested showed an increase in the Ct-values. Nevertheless, the sensitivity of the test was not affected. Thus, the test for Schmallenberg virus detection is enabled for the possible emergency of this agent in Cuba.
Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic w... more Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of the...
To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba... more To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.
The presence of infection by human T cell lymphotropic virus type 1 (HTLV-1) in Cuba has been pre... more The presence of infection by human T cell lymphotropic virus type 1 (HTLV-1) in Cuba has been previously documented. However, genetic information on the strains that circulate in the Cuban people remains unknown. The present work constitutes the first study of phylogenetic relationship of HTLV-1 Cuban isolates. Twelve Cuban patients who were diagnosed with HTLV-1 infection and had different clinical manifestations were studied. The 3' LTR sequences were analyzed for the construction of a phylogenetic tree with reference sequences of HTLV-1 of different geographic origins. Phylogenetic analysis of the 3' LTR gene showed that all the Cuban samples clustered in the Transcontinental subgroup of the Cosmopolitan subtype. Phylogenetic analysis suggests multiple introductions of HTLV-1 in Cuba as well as a possible African origin of the samples. The results of the study will reinforce the program of epidemic surveillance of the infection in Cuba.
The emergence of the pandemic H1N1/2009 influenza virus poses a potential global threat for human... more The emergence of the pandemic H1N1/2009 influenza virus poses a potential global threat for human and animal health. In this study, we carried out pandemic H1N1/2009 influenza virus surveillance in swine herds in Cuba intending to determine whether the virus was circulating among pig populations. As a result we describe, for the first time, the detection of pandemic H1N1/2009 influenza virus in swine herds in Cuba. In addition, phylogenetic analysis and molecular characterization of three viral isolates were performed. Phylogenetic relationships confirmed that all of the eight genes of the three isolates were derived from the pandemic H1N1/2009 virus. The Cuban isolates, formed an independent cluster within the pandemic H1N1/2009 influenza strains. Different molecular markers, previously described in pandemic H1N1/2009 influenza viruses, related with adaptive evolution, viral evasion from the host-immune response, virulence and dissemination were also present in Cuban pandemic H1N1/2009 isolates.
In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each ye... more In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. Interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. This study aimed to assess positive selection pressure acting on partial E2 gene of CSF viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (CSFV) evolution in swine populations under regular vaccination. Selection pressure analysis were performed to detect positive selection acting on a particular lineage as well as among sites of the E2-B/C-domain of CSFV nucleotide sequences, reported in a previous study and in the present work, several models, available in the CODEML module of PAML 4.3, were used. In addition, a representative Cuban CSF isolate was assessed in an experimental infection trial for their clinical virulence in order to expand the knowledge regarding CSF viruses circulating in pig populations. The viral genomes sequenced in this study were grouped in a defined cluster within the genotype 1.2, as it has been reported previously for Cuban CSF viruses. The selection pressure analysis didn't find evidence of positive selection (dN/dS of > 1) along any branch. The positive selective pressure analysis estimated six new sites under positive selection on E2 partial gene analysed. Besides, the clinical manifestations of the CSF-disease were related mainly to a mild course of the illness. The high number of positively selected sites suggests that these changes could be associated to viral evasion of the host-immune response. These observations highlight a possible association between escape viral variants and the alterations observed in the virulence and pathogenesis of the virus. Therefore, while the vaccination programs have not led to a genotype change, alterations in virulence were suggested to arise.
Multiple viral infections are common in pigs under intensive production conditions. All five of t... more Multiple viral infections are common in pigs under intensive production conditions. All five of the viruses included in this study are associated with multifactorial diseases that cause significant economic losses in swine farming worldwide. The development is described of a novel multiple real-time PCR system based on the use of SYBR Green I that allows the simultaneous detection and differentiation of porcine circovirus 2 (PCV-2), porcine parvovirus (PPV), pseudorabies virus (PRV) and Torque teno sus virus species 1 and 2 (TTSuV1 and TTSuV2) in pigs. The method was able to distinguish between all five viral agents, and tests of other DNA viruses proved the specificity of the system. The multiple real-time PCR system was sensitive, as the limits of detection ranged from 3.65×10(3) to 5.04×10(3) copies of DNA template per reaction. The coefficients of variation were low for both intra-assay and inter-assay variability. In addition, the results of the multiple real-time PCR system tests were 100% consistent with previous results based on specific PCR assay testing of field samples. This method could be a useful tool for epidemiological studies and disease management.
Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic w... more Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred.
In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screene... more In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screened by PCR for the presence of TTSuV1, TTSuV2, PCV-2, PPV and CSFV in spleen samples. The variability of the porcine TTSuV sequences obtained was investigated by phylogenetic analysis. This study showed for the first time that TTSuV1 and TTSuV2 were present in Cuban swine herds. The investigation revealed the following infection rates: TTSuV1 40%, TTSuV2 37.5%, PCV-2 70%, PPV 37.5% and CSFV in 52.5%. The presence of two or more of these viruses at different rates in the same spleen samples was revealed. Also, a higher genetic diversity of TTSuV2 sequences was observed regarding TTSuV1 sequences.
Many methods are used to detect antibiotic resistance genes in samples. The objective of the stud... more Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.
Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulte... more Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of ...
Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused ... more Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. The current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment B. This marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent IBD virus (vvIBDV) strains worldwide. Sequences of the segment B gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank Database; Cuban sequences were obtained in the current work. A phylogenetic marker named B-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. This last parameter is based on the ability of B-marker to reconstruct the same topology as the complete segment B of the viral genome. From the results obtained from B-marker, demographic history for both main lineages of IBDV regarding segment B was performed by Bayesian skyline plot analysis. Phylogenetic analysis for both segments of IBDV genome was also performed, revealing the presence of a natural reassortant strain with segment A from vvIBDV strains and segment B from non-vvIBDV strains within Cuban IBDV population. This study contributes to a better understanding of the emergence of vvIBDV strains, describing molecular epidemiology of IBDV using the state-of-the-art methodology concerning phylogenetic reconstruction. This study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvIBDV strains. Therefore, it highlights the need to obtain information about both genome segments of IBDV for molecular epidemiology studies.
Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic d... more Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection.
Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronch... more Infectious bronchitis is a highly contagious viral disease of poultry caused by infectious bronchitis virus (IBV) and is considered one of the most economically important viral diseases of chickens. Control of IBV has been attempted using live attenuated and inactivated vaccines. Live attenuated vaccines of the Massachusetts (Mass.) serotype are the most commonly used for this purpose. Due to the continuous emergence of new variants of the infectious bronchitis virus, the identification of the type of IBV causing an outbreak in commercial poultry is important in the selection of the appropriate vaccine(s) capable of inducing a protective immune response. The present work was aimed at developing and evaluating a duplex SYBR Green I-based real-time RT-PCR (rRT-PCR) assay for the simultaneous detection and differentiation of Mass. and non-Mass. serotypes of IBV. The duplex rRT-PCR yielded curves of amplification with two specific melting curves (Tm1 ¼ 83 C AE 0.5 C and Tm2 ¼ 87 C AE 0.5 C) and only one specific melting peak (Tm ¼ 87 C AE 0.5 C) when the IBV Mass. serotype and IBV non-Mass. serotype strains were evaluated, respectively. The detection limit of the assay was 8.2 gene copies/mL based on in vitro transcribed RNA and 0.1 EID 50 /mL. The assay was able to detect all the IBV strains assessed and discriminated well among the IBV Mass. and the IBV non-Mass. serotypes strains. In addition, amplification curves were not obtained with any of the other viruses tested. From the 300 field samples tested, the duplex rRT-PCR yielded a total of 80 samples that were positive for IBV (26.67%), 73 samples identified as the IBV Mass. serotype and seven samples as identified as the IBV non-Mass. serotype. A comparison of the performance of test as assessed with field samples revealed that the duplex rRT-PCR detected a higher number of IBV-positive samples than when conventional RT-PCR or virus isolation tests were used. The duplex rRT-PCR presented here is a useful tool for the rapid identification of outbreaks and for surveillance programmes during IB-suspected cases, particularly in countries with a vaccination control programme.
Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of... more Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of the most important diseases in swine. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. Viral miRNAs have recently been described and the number of viral miRNAs has been increasing in the past few years. In this study, small RNA libraries were constructed from two tissues of subclinically PCV2 infected pigs to explore if PCV2 can encode viral miRNAs. The deep sequencing data revealed that PCV2 does not express miRNAs in an in vivo subclinical infection.
Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adul... more Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.
In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated fr... more In this report, we describe the emergence of reassorted H1N1 swine influenza virus, originated from a reassortment event between the H1N1 pandemic influenza virus (H1N1p/2009) and endemic swine influenza virus in Cuban swine population. In November 2010, a clinical respiratory outbreak was reported on a pig fattening farm in Cuba. Phylogenetic analysis showed that all the genes of one of the isolate obtained, with the exception of neuraminidase, belonged to the H1N1p/2009 cluster. This finding suggests that H1N1pdm has been established in swine and has become a reservoir of reassortment that may produce new viruses with both animal and public health risks.
Background: Infectious bursal disease is a highly contagious and acute viral disease caused by th... more Background: Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains.
Schmallenberg virus was first detected in Germany in October 2011, associated with congenital mal... more Schmallenberg virus was first detected in Germany in October 2011, associated with congenital malformations in cattle, sheep and goats. This novel emergent agent causes mild disease in cattle with decreased milk production, fever and diarrhea. In March 2012, the German Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, reported the development and validation of a real-time RT-PCR for the diagnosis of this new virus. The Animal Virology Laboratory at the National Center for Animal and Plant Health in Cuba has adapted the protocol previously reported on the LightCycler platforms using two different mix conditions. In all cases, amplification curves obtained were specific and all the dilutions tested showed an increase in the Ct-values. Nevertheless, the sensitivity of the test was not affected. Thus, the test for Schmallenberg virus detection is enabled for the possible emergency of this agent in Cuba.
Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic w... more Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of the...
To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba... more To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.
The presence of infection by human T cell lymphotropic virus type 1 (HTLV-1) in Cuba has been pre... more The presence of infection by human T cell lymphotropic virus type 1 (HTLV-1) in Cuba has been previously documented. However, genetic information on the strains that circulate in the Cuban people remains unknown. The present work constitutes the first study of phylogenetic relationship of HTLV-1 Cuban isolates. Twelve Cuban patients who were diagnosed with HTLV-1 infection and had different clinical manifestations were studied. The 3' LTR sequences were analyzed for the construction of a phylogenetic tree with reference sequences of HTLV-1 of different geographic origins. Phylogenetic analysis of the 3' LTR gene showed that all the Cuban samples clustered in the Transcontinental subgroup of the Cosmopolitan subtype. Phylogenetic analysis suggests multiple introductions of HTLV-1 in Cuba as well as a possible African origin of the samples. The results of the study will reinforce the program of epidemic surveillance of the infection in Cuba.
The emergence of the pandemic H1N1/2009 influenza virus poses a potential global threat for human... more The emergence of the pandemic H1N1/2009 influenza virus poses a potential global threat for human and animal health. In this study, we carried out pandemic H1N1/2009 influenza virus surveillance in swine herds in Cuba intending to determine whether the virus was circulating among pig populations. As a result we describe, for the first time, the detection of pandemic H1N1/2009 influenza virus in swine herds in Cuba. In addition, phylogenetic analysis and molecular characterization of three viral isolates were performed. Phylogenetic relationships confirmed that all of the eight genes of the three isolates were derived from the pandemic H1N1/2009 virus. The Cuban isolates, formed an independent cluster within the pandemic H1N1/2009 influenza strains. Different molecular markers, previously described in pandemic H1N1/2009 influenza viruses, related with adaptive evolution, viral evasion from the host-immune response, virulence and dissemination were also present in Cuban pandemic H1N1/2009 isolates.
In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each ye... more In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. Interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. This study aimed to assess positive selection pressure acting on partial E2 gene of CSF viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (CSFV) evolution in swine populations under regular vaccination. Selection pressure analysis were performed to detect positive selection acting on a particular lineage as well as among sites of the E2-B/C-domain of CSFV nucleotide sequences, reported in a previous study and in the present work, several models, available in the CODEML module of PAML 4.3, were used. In addition, a representative Cuban CSF isolate was assessed in an experimental infection trial for their clinical virulence in order to expand the knowledge regarding CSF viruses circulating in pig populations. The viral genomes sequenced in this study were grouped in a defined cluster within the genotype 1.2, as it has been reported previously for Cuban CSF viruses. The selection pressure analysis didn't find evidence of positive selection (dN/dS of > 1) along any branch. The positive selective pressure analysis estimated six new sites under positive selection on E2 partial gene analysed. Besides, the clinical manifestations of the CSF-disease were related mainly to a mild course of the illness. The high number of positively selected sites suggests that these changes could be associated to viral evasion of the host-immune response. These observations highlight a possible association between escape viral variants and the alterations observed in the virulence and pathogenesis of the virus. Therefore, while the vaccination programs have not led to a genotype change, alterations in virulence were suggested to arise.
Multiple viral infections are common in pigs under intensive production conditions. All five of t... more Multiple viral infections are common in pigs under intensive production conditions. All five of the viruses included in this study are associated with multifactorial diseases that cause significant economic losses in swine farming worldwide. The development is described of a novel multiple real-time PCR system based on the use of SYBR Green I that allows the simultaneous detection and differentiation of porcine circovirus 2 (PCV-2), porcine parvovirus (PPV), pseudorabies virus (PRV) and Torque teno sus virus species 1 and 2 (TTSuV1 and TTSuV2) in pigs. The method was able to distinguish between all five viral agents, and tests of other DNA viruses proved the specificity of the system. The multiple real-time PCR system was sensitive, as the limits of detection ranged from 3.65×10(3) to 5.04×10(3) copies of DNA template per reaction. The coefficients of variation were low for both intra-assay and inter-assay variability. In addition, the results of the multiple real-time PCR system tests were 100% consistent with previous results based on specific PCR assay testing of field samples. This method could be a useful tool for epidemiological studies and disease management.
Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic w... more Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred.
In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screene... more In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screened by PCR for the presence of TTSuV1, TTSuV2, PCV-2, PPV and CSFV in spleen samples. The variability of the porcine TTSuV sequences obtained was investigated by phylogenetic analysis. This study showed for the first time that TTSuV1 and TTSuV2 were present in Cuban swine herds. The investigation revealed the following infection rates: TTSuV1 40%, TTSuV2 37.5%, PCV-2 70%, PPV 37.5% and CSFV in 52.5%. The presence of two or more of these viruses at different rates in the same spleen samples was revealed. Also, a higher genetic diversity of TTSuV2 sequences was observed regarding TTSuV1 sequences.
Many methods are used to detect antibiotic resistance genes in samples. The objective of the stud... more Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.
Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulte... more Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of ...
Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused ... more Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. The current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment B. This marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent IBD virus (vvIBDV) strains worldwide. Sequences of the segment B gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank Database; Cuban sequences were obtained in the current work. A phylogenetic marker named B-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. This last parameter is based on the ability of B-marker to reconstruct the same topology as the complete segment B of the viral genome. From the results obtained from B-marker, demographic history for both main lineages of IBDV regarding segment B was performed by Bayesian skyline plot analysis. Phylogenetic analysis for both segments of IBDV genome was also performed, revealing the presence of a natural reassortant strain with segment A from vvIBDV strains and segment B from non-vvIBDV strains within Cuban IBDV population. This study contributes to a better understanding of the emergence of vvIBDV strains, describing molecular epidemiology of IBDV using the state-of-the-art methodology concerning phylogenetic reconstruction. This study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvIBDV strains. Therefore, it highlights the need to obtain information about both genome segments of IBDV for molecular epidemiology studies.
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Papers by Lester Pérez