Molecular Epidemiology of Rotavirus A in Calves: Evolutionary Analysis of a Bovine G8P[11] Strain and Spatio-Temporal Dynamics of G6 Lineages in the Americas.

Rotavirus A (RVA) causes diarrhea in calves and frequently possesses the G6 and P[5]/P[11] genotypes, whereas G8 is less common. We aimed to compare RVA infections and G/P genotypes in beef and dairy calves from major livestock regions of Argentina, elucidate the evolutionary origin of a G8 strain and analyze the G8 lineages, infer the phylogenetic relationship of RVA field strains, and investigate the evolution and spatio-temporal dynamics of the main G6 lineages in American countries. Fecal samples (n = 422) from diarrheic (beef, 104; dairy, 137) and non-diarrheic (beef, 78; dairy, 103) calves were analyzed by ELISA and semi-nested multiplex RT-PCR. Sequencing, phylogenetic, phylodynamic, and phylogeographic analyses were performed. RVA infections were more frequent in beef (22.0%) than in dairy (14.2%) calves. Prevalent genotypes and G6 lineages were G6(IV)P[5] in beef (90.9%) and G6(III)P[11] (41.2%) or mixed genotypes (23.5%) in dairy calves. The only G8 strain was phylogenetically related to bovine and artiodactyl bovine-like strains. Re-analyses inside the G8 genotype identified G8(I) to G8(VIII) lineages. Of all G6 strains characterized, the G6(IV)P[5](I) strains from "Cuenca del Salado" (Argentina) and Uruguay clustered together. According to farm location, a clustering pattern for G6(IV)P[5] strains of beef farms was observed. Both G6 lineage strains together revealed an evolutionary rate of 1.24 × 10-3 substitutions/site/year, and the time to the most recent common ancestor was dated in 1853. The most probable ancestral locations were Argentina in 1981 for G6(III) strains and the USA in 1940 for G6(IV) strains. The highest migration rates for both G6 lineages together were from Argentina to Brazil and Uruguay. Altogether, the epidemiology, genetic diversity, and phylogeny of RVA in calves can differ according to the production system and farm location. We provide novel knowledge about the evolutionary origin of a bovine G8P[11] strain. Finally, bovine G6 strains from American countries would have originated in the USA nearly a century before its first description.

Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages' strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.

An Unwanted Association: The Threat to Papaya Crops by a Novel Potexvirus in Northwest Argentina.

An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.

Molecular characterization of group A rotavirus strains detected in alpacas (Vicugna pacos) from Peru.

The alpaca is a very important social and economic resource for the production of fibre and meat for Andean communities. Peru is the main producer of alpacas. Group A rotavirus (RVA) has been sporadically detected in alpacas. In this study, a total of 1423 faecal samples from alpacas from different locations of the Puno department in Peru were collected and analysed by an antigen-capture ELISA in order to detect RVA. Four per cent of the samples were RVA-positive (57/1423). The genotype constellation of three selected alpaca RVA strains were G3/8 P[1/14]-I2-R2/5-C2/3-M2/3-A17-N2/3-T6-E3-H3. Two of the analysed strains presented a bovine-like genotype constellation, whereas the third strain presented six segments belonging to the AU-1-like genogroup (G3, M3, C3, N3, T3 and E3), suggesting reassorting events. Monitoring of the sanitary health of juvenile alpacas is essential to reduce the rates of neonatal mortality and for the development of preventive health strategies.

Molecular characterization of yerba mate chlorosis-associated virus, a putative cytorhabdovirus infecting yerba mate (Ilex paraguariensis).

We present the molecular characterization of a new virus infecting yerba mate (Ilex paraguariensis St. Hil.) in Argentina. Deep sequencing of diseased yerba mate plants showing chlorotic linear patterns, chlorotic rings, and vein yellowing resulted in the identification of a new virus resembling plant rhabdoviruses in sequence and genome structure. We have determined the complete genome sequence of this virus, which is 12,876 nt long. Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-P4-M-G-L-5'. Phylogenetic analysis suggested that the described virus is a new member of the genus Cytorhabdovirus, which was supported by the observation of rhabdovirus-like particles within the cytoplasm of infected yerba mate cells. The virus has been tentatively named "yerba mate chlorosis-associated virus" (YmCaV). The availability of the YmCaV genome sequence will contribute to assessing the genetic variability of this virus and determining its role in this yerba mate disease.

Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America.

Unusual chromatin state in Rhynchosciara americana (Diptera: Sciaridae).

Euchromatin and heterochromatin are usually defined by the degree of DNA compaction, gene content and combinations of histone and non-histone proteins. More recent studies on protein location have been able to specify a variety of chromatin types thus adding chromatin configurations other than the two basic reference states. Chromatin research exploiting non-model organisms has the potential to provide novel information related to epigenetic modifications and their impact on chromosome structure and function. Polytene chromosomes of Rhynchosciara americana display a particular region within the A9 sub-section characterised by lack of DNA compaction as well as an usual polytene banding pattern. DNA content in the sub-section seems to be low as deduced by DAPI staining. Antibodies to H3K4me, a conserved epigenetic transcription marker,labelled the A9 sub-section strongly. In contrast,transcriptional activity in the region, if any, seems to be low as inferred by detection of RNA polymerase II and RNA. Histone markers related to heterochromatin formation such as H3K9me and H3K27me are underrepresented in the A9 sub-section. However, a chromodomain-containing sciarid protein was detected in the region, displaying levels of fluorescence very close to those observed in pericentric heterochromatin.A plasmid micro-library constructed with microdissected DNA from the A9 sub-section was screened for repetitive DNA. The proportion of inserts containing repeats was found to be similar to that contained in another micro-library made with DNA from a single chromosome end of this species. The data suggest an unusual "chromatin colour" indicating that high levels of histone markers related to transcription coexist with a significant presence of chromodomain-containing proteins and the virtual absence of histone modifications observed in heterochromatin formation.

ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: a new blaTEM gene.

Resistance to ß-lactam/ß-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV ß-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8µg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum ß-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: A new blaTEMgene / ß-lactamasas producidas por enterobacterias resistentes a amoxicilina-ácido clavulánico aisladas en Buenos Aires, Argentina: un nuevo gen blaTEM

La resistencia a la combinación de ß-lactámico/inhibidor de ß-lactamasa en enterobacterias es un problema creciente que no ha sido estudiado intensamente en Argentina. En el presente trabajo, 54/843 enterobacterias recolectadas en un hospital universitario de la ciudad de Buenos Aires fueron resistentes a ampicilina-sulbactama, pero se mantuvieron sensibles a las cefalosporinas de segunda y tercera generación. Se analizaron los mecanismos enzimáticos presentes en los aislamientos que también fueron resistentes a amoxicilina-ácido clavulánico (AMC) (18/54). La secuenciación reveló dos variantes diferentes de blaTEM-1, donde blaTEM-1b es el alelo más frecuentemente detectado (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis y 1 Raoultella terrigena), seguidos por blaTEM-1a(1 K. pneumoniae). La resistencia a AMC parece estar asociada principalmente con la hiperproducción de TEM-1 (sobre todo en E. coli) o con la coexpresión con ß-lactamasas tipo OXA-2 y/o SHV (K. pneumoniae y P. mirabilis). Se describió una nueva variante de blaTEM(TEM-163) en un aislamiento de E. coli que presentó una CIM frente a AMC de 16/8 µg/ml. La enzima TEM-163 contiene dos sustituciones de aminoácidos respecto de TEM-1, Arg275Gln y His289Leu. Teniendo en cuenta la alta actividad específica observada y la baja IC50 para el ácido clavulánico, el patrón de resistencia de este aislamiento parece obedecer a la hiperproducción de la nueva variante de la ß-lactamasa de amplio espectro, en lugar de vincularse con un comportamiento similar al de una TEM resistente a inhibidores (IRT).

Resistance to ß-lactam/ß-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second-and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a(1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV ß-lactamases (K. pneumoniae and P. mirabilis). A new blaTEMvariant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8 µg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrumß-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: A new blaTEMgene / ß-lactamasas producidas por enterobacterias resistentes a amoxicilina-ácido clavulánico aisladas en Buenos Aires, Argentina: un nuevo gen blaTEM

La resistencia a la combinación de ß-lactámico/inhibidor de ß-lactamasa en enterobacterias es un problema creciente que no ha sido estudiado intensamente en Argentina. En el presente trabajo, 54/843 enterobacterias recolectadas en un hospital universitario de la ciudad de Buenos Aires fueron resistentes a ampicilina-sulbactama, pero se mantuvieron sensibles a las cefalosporinas de segunda y tercera generación. Se analizaron los mecanismos enzimáticos presentes en los aislamientos que también fueron resistentes a amoxicilina-ácido clavulánico (AMC) (18/54). La secuenciación reveló dos variantes diferentes de blaTEM-1, donde blaTEM-1b es el alelo más frecuentemente detectado (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis y 1 Raoultella terrigena), seguidos por blaTEM-1a(1 K. pneumoniae). La resistencia a AMC parece estar asociada principalmente con la hiperproducción de TEM-1 (sobre todo en E. coli) o con la coexpresión con ß-lactamasas tipo OXA-2 y/o SHV (K. pneumoniae y P. mirabilis). Se describió una nueva variante de blaTEM(TEM-163) en un aislamiento de E. coli que presentó una CIM frente a AMC de 16/8 Ag/ml. La enzima TEM-163 contiene dos sustituciones de aminoácidos respecto de TEM-1, Arg275Gln y His289Leu. Teniendo en cuenta la alta actividad específica observada y la baja IC50 para el ácido clavulánico, el patrón de resistencia de este aislamiento parece obedecer a la hiperproducción de la nueva variante de la ß-lactamasa de amplio espectro, en lugar de vincularse con un comportamiento similar al de una TEM resistente a inhibidores (IRT).(AU)

Resistance to ß-lactam/ß-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second-and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a(1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV ß-lactamases (K. pneumoniae and P. mirabilis). A new blaTEMvariant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8 Ag/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrumß-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.(AU)

The first caprine rotavirus detected in Argentina displays genomic features resembling virus strains infecting members of the Bovidae and Camelidae.

Rotavirus group A (RVA) is a major cause of diarrhea in humans and young animals including small ruminants. The purpose of this study was to identify RVA in dairy goat kids, and to characterize the complete genomic constellation and genetic relatedness with other RVA strains. Four out of twenty fecal samples from diarrheic and non-diarrheic goat kids were positive for RVA by ELISA. A representative sample was selected for further genome analyses. The RVA strain RVA/Goat-wt/ARG/0040/2011/G8P[1] displayed the following genomic constellation: G8-P[1]-I2-R5-C2-M2-A3-N2-T6-E12-H3, reminiscent to guanaco and other bovine-like RVA strains detected in Argentina. Phylogenetic analyses revealed that most of the genome segments had a rather close relatedness with RVA strains typically obtained from cattle, sheep, South American camelids and goats. Interestingly, strain 0040 possessed the R5 and E12 genotypes which have up to date only been found in different animal species from Argentina. Overall, these findings suggest that strain 0040 could represent a typical goat RVA genome constellation similar to those previously found in other animal species within the order Artiodactyla.

ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: a new blaTEM gene. / ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: a new blaTEM gene.

Resistance to ß-lactam/ß-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV ß-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8Ag/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum ß-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

Discovery and molecular characterization of a group A rotavirus strain detected in an Argentinean vicuña (Vicugna vicugna).

The wild vicuña (Vicugna vicugna) is one of the four species of native South American camelids (SACs) in addition to the wild guanaco, and their domesticated counterparts, alpaca and llama, respectively. Serological data have indicated the presence of group A rotaviruses (RVA) specific antibodies in all 4 members of the SAC, and so far, RVA has been detected from alpacas, llamas and guanacos. A total of 59 fecal samples from healthy wild newborn and juvenile vicuñas, raised in captivity in Jujuy, Argentina were collected and analyzed by ELISA to detect RVA antigen. Two samples (3%) were found to contain G8 RVA strains and one strain (RVA/Vicuña-wt/ARG/C75/2010/G8P[14]) was selected for further genome analyses, revealing the G8-P[14]-I2-R2-C2-M2-Ax-N2-T6-E3-Hx genotype constellation. Unfortunately, no sequence data could be obtained for NSP1 and NSP5. Except for the E3 NSP4 genotype, this partial genotype constellation is reminiscent to bovine RVA strains and bovine-like RVA strains isolated from sheep, guanaco, antelope and humans. This relationship was confirmed phylogenetically, providing further evidence of the widespread presence of this genotype constellation in animals belonging to the artiodactyls. In particular, a close phylogenetic relationship was found between C75 and guanaco RVA strain RVA/Guanaco-wt/ARG/Chubut/1999/G8P[14] for at least 5 gene segments, suggesting a partial conservation of the genotype constellation of RVA strains infecting different species of SACs, even though nowadays their natural habitats are not overlapping. The further monitoring of the sanitary health of wild newborn and juvenile vicuñas is essential to improve the management practices applied in their sustainable exploitation.

Complete molecular genome analyses of equine rotavirus A strains from different continents reveal several novel genotypes and a largely conserved genotype constellation.

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to non-equine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.