Co-Circulation of Multiple Coronavirus Genera and Subgenera during an Epizootic of Lethal Respiratory Disease in Newborn Alpacas (Vicugna pacos) in Peru: First Report of Bat-like Coronaviruses in Alpacas.

Coronaviruses (CoVs) infect a wide range of hosts, including humans, domestic animals, and wildlife, typically causing mild-to-severe respiratory or enteric disease. The main objective of this study was to identify CoV genera and subgenera detected in Peruvian alpacas. Lung lavage specimens were collected from 32 animals aged 1 to 6 weeks. CoVs were identified by using RT-PCR to amplify a pan-CoV conserved region of the RNA-dependent RNA polymerase-encoding gene. A nested PCR was performed to identify ß-CoVs. Then, ß-CoV-positive samples were subjected to RT-PCR using specific primers to identify the Embecovirus subgenus. Out of 32 analyzed samples, 30 (93.8%) tested positive for at least one CoV genus. ß-, α-, or unclassified CoVs were identified in 24 (80%), 1 (3.3%), and 1 (3.3%) of the positive samples, respectively. A CoV genus could not be identified in two (6.7%) samples. A mixture of different CoV genera was detected in two (6.7%) samples: one was co-infected with ß- and α-CoVs, and the other contained a ß- and an unclassified CoV. A sequence analysis of the amplicons generated by the PCR identified 17 ß-CoV strains belonging to the subgenus Embecovirus and two α-CoV strains belonging to Decacovirus. A phylogenetic analysis of two strains revealed a relationship with an unclassified Megaderma BatCoV strain. A subgenus could not be identified in nine ß-CoV samples. Our data show a high prevalence and a high genetic diversity of CoV genera and subgenera that infect alpacas, in which the ß-CoV subgenus Embecovirus predominated. Our data also suggest a new role for bats in the dissemination and transmission of uncommon CoVs to alpacas raised in rural Peru.

Stability of ACE2 Peptide Mimetics and Their Implications on the Application for SARS-CoV2 Detection.

The SARS-CoV-2 worldwide outbreak prompted the development of several tools to detect and treat the disease. Among the new detection proposals, the use of peptides mimetics has surged as an alternative to avoid the use of antibodies, of which there has been a shortage during the COVID-19 pandemic. However, the use of peptides in detection systems still presents some questions to be answered, mainly referring to their stability under different environmental conditions. In this work, we synthesized an ACE2 peptide mimic and evaluated its stability in different pH, salinity, polarity, and temperature conditions. Further, the same conditions were assessed when using the ability of the peptide mimic to detect the recombinant SARS-CoV-2 spike protein in a biotin-streptavidin-enzyme-linked assay. Finally, we also tested the capacity of the peptide to detect SARS-CoV-2 from patients' samples. The results indicate that the peptide is structurally sensitive to the medium conditions, with relevance to the pH, where basic pH favored its performance when used as a SARS-CoV-2 detector. Further, the proposed peptide mimic was able to detect SARS-CoV-2 comparably to RT-qPCR results. Therefore, the present study promotes knowledge advancement, particularly in terms of stability considerations, in the application of peptide mimics as a replacement for antibodies in detection systems.

Enteric Viral Infections among Domesticated South American Camelids: First Detection of Mammalian Orthoreovirus in Camelids.

Enteric infections are a major cause of neonatal death in South American camelids (SACs). The aim of this study was to determine the prevalence of enteric viral pathogens among alpacas and llamas in Canchis, Cuzco, located in the southern Peruvian highland. Fecal samples were obtained from 80 neonatal alpacas and llamas and tested for coronavirus (CoV), mammalian orthoreovirus (MRV), and rotavirus A (RVA) by RT-PCR. Of the 80 fecal samples analyzed, 76 (95%) were positive for at least one of the viruses tested. Overall, the frequencies of positive samples were 94.1% and 100% among alpacas and llamas, respectively. Of the positive samples, 33 (43.4%) were monoinfected, while 43 (56.6%) had coinfections with two (83.7%) or three (16.3%) viruses. CoV was the most commonly detected virus (87.5%) followed by MRV (50%). RVA was detected only in coinfections. To our knowledge, this is the first description of MRV circulation in SACs or camelids anywhere. These data show that multiple viruses circulate widely among young alpaca and llama crias within the studied areas. These infections can potentially reduce livestock productivity, which translates into serious economic losses for rural communities, directly impacting their livelihoods.

[Detection of the ermB gene associated with macrolide resistance in Campylobacter strains isolated from chickens marketed in Lima, PeruDetecção do gene ermB associado à resistência a macrolídeos em cepas de Campylobacter isoladas de frangos comercializados em Lima, no Peru]. / Detección del gen ermB asociado a la resistencia a macrólidos en cepas de Campylobacter aisladas de pollos comercializados en Lima, Perú.

OBJECTIVE: To detect the presence of the ermB gene associated with macrolide resistance in Campylobacter spp. strains isolated from chickens marketed in Lima, Peru. METHODS: 120 samples of chicken skin from three markets in the districts of San Martin de Porres (n = 30), Santa Anita (n = 20), and Independencia (n = 70), located in the Province of Lima, Peru, were analyzed. Microbiological analysis of the samples was carried out according to ISO standard 10272-1:2017. For the polymerase chain reaction (PCR) confirmation of genus and species, 16-rRNA and GlyA and hipO primers, respectively, were used. For the evaluation of antibiotic sensitivity, the Müller-Hinton agar with 5% blood, with sensi-discs for azithromycin (15 µg) and erythromycin (15 µg), was used. For detection of the ermB gene in strains with resistant phenotypes, conventional PCR was used. RESULTS: A total of 117 positive samples (97.5%) were obtained; of these, 100% were compatible with Campylobacter coli (negative hippurate test) and confirmed by PCR. The plate-based assessment of antibiotic resistance to azithromycin and erythromycin resulted in 100% of strains with a phenotype that is resistant to these macrolides, while the PCR to detect the ermB gene indicated a total of 62 positives (53%), which were confirmed through sequencing. CONCLUSIONS: These results demonstrate that the chicken carcasses sold in markets in Lima present contamination by C. coli with high resistance to macrolides, which can be attributed to the presence of the ermB gene.

OBJETIVO: Detectar a presença do gene ermB associado à resistência a macrolídeos em cepas de Campylobacter spp. isoladas de frangos comercializados em Lima, no Peru. MÉTODOS: Analisamos 120 amostras de pele de frango provenientes de três mercados nos distritos de San Martín de Porres (n=30), Santa Anita (n=20) e Independencia (n=70), situados na Província de Lima, no Peru. Realizamos uma análise microbiológica das amostras de acordo com as recomendações da norma ISO 10272-1:2017. Para a confirmação do gênero e espécie por reação em cadeia da polimerase (PCR), utilizamos os primers 16-rRNA, GlyA e hypO. Para avaliar a sensibilidade antimicrobiana, utilizamos ágar de Müller-Hinton-sangue a 5% com discos de sensibilidade de azitromicina (15 µg) e eritromicina (15 µg). A detecção do gene ermB em cepas com fenótipos resistentes foi feita por PCR convencional. RESULTADOS: Obtivemos um total de 117 amostras positivas (97,5%), das quais 100% foram compatíveis com Campylobacter coli (teste do hipurato negativo) e confirmadas por PCR. Na avaliação da resistência antimicrobiana em placa para azitromicina e eritromicina, 100% das cepas apresentaram fenótipo de resistência a estes macrolídeos, enquanto a PCR para a detecção do gene ermB indicou um total de 62 cepas positivas (53%), que foram confirmadas por sequenciamento. CONCLUSÕES: Estes resultados demonstram que as carcaças de frango comercializadas nos mercados de Lima apresentam contaminação por C. coli com alta resistência a macrolídeos, o que pode ser atribuído à presença do gene ermB.

Detección del gen ermB asociado a la resistencia a macrólidos en cepas de Campylobacter aisladas de pollos comercializados en Lima, Perú

[RESUMEN]. Objetivo. Detectar la presencia del gen ermB asociado a la resistencia a macrólidos en cepas de Campylobacter spp. aisladas de pollos comercializados en Lima, Perú. Métodos. Se analizaron 120 muestras de piel de pollo provenientes de tres mercados de los distritos de San Martín de Porres (n = 30), Santa Anita (n = 20) e Independencia (n = 70), ubicados en la Provincia de Lima, Perú. Se realizó el análisis microbiológico de las muestras según las recomendaciones de la norma ISO 10272-1:2017. Para la confirmación por reacción en cadena de la polimerasa (PCR, por sus siglas en inglés) de género y especie, se utilizaron los cebadores (primers) 16-rARN y GlyA e hipO, respectivamente. Para evaluar la sensibilidad antibiótica, se utilizó el agar Müller-Hinton sangre al 5% con sensidiscos de azitromicina (15 μg) y eritromicina (15 μg). La detección del gen ermB en cepas con fenotipos resistentes se realizó mediante PCR convencional. Resultados. Se obtuvo un total de 117 muestras positivas (97,5%), de las cuales 100% fueron compatibles con Campylobacter coli (prueba de hipurato negativa) y confirmadas por PCR. La evaluación de resistencia antibiótica en placa para azitromicina y eritromicina dio como resultado 100% de cepas con fenotipo de resistencia a estos macrólidos, mientras que la PCR para la detección del gen ermB indicó un total de 62 positivas (53%), que fueron confirmadas por secuenciamiento. Conclusiones. Estos resultados demuestran que las carcasas de pollo comercializadas en mercados de Lima presentan contaminación por C. coli con una alta resistencia a macrólidos, lo cual puede ser atribuido a la presencia del gen ermB.

[ABSTRACT]. Objective. To detect the presence of the ermB gene associated with macrolide resistance in Campylobacter spp. strains isolated from chickens marketed in Lima, Peru. Methods. 120 samples of chicken skin from three markets in the districts of San Martin de Porres (n = 30), Santa Anita (n = 20), and Independencia (n = 70), located in the Province of Lima, Peru, were analyzed. Microbiological analysis of the samples was carried out according to ISO standard 10272-1:2017. For the polymerase chain reaction (PCR) confirmation of genus and species, 16-rRNA and GlyA and hipO primers, respectively, were used. For the evaluation of antibiotic sensitivity, the Müller-Hinton agar with 5% blood, with sensi-discs for azithromycin (15 μg) and erythromycin (15 μg), was used. For detection of the ermB gene in strains with resistant phenotypes, conventional PCR was used. Results. A total of 117 positive samples (97.5%) were obtained; of these, 100% were compatible with Campylobacter coli (negative hippurate test) and confirmed by PCR. The plate-based assessment of antibiotic resistance to azithromycin and erythromycin resulted in 100% of strains with a phenotype that is resistant to these macrolides, while the PCR to detect the ermB gene indicated a total of 62 positives (53%), which were confirmed through sequencing. Conclusions. These results demonstrate that the chicken carcasses sold in markets in Lima present contamination by C. coli with high resistance to macrolides, which can be attributed to the presence of the ermB gene.

[RESUMO]. Objetivo. Detectar a presença do gene ermB associado à resistência a macrolídeos em cepas de Campylobacter spp. isoladas de frangos comercializados em Lima, no Peru. Métodos. Analisamos 120 amostras de pele de frango provenientes de três mercados nos distritos de San Martín de Porres (n=30), Santa Anita (n=20) e Independencia (n=70), situados na Província de Lima, no Peru. Realizamos uma análise microbiológica das amostras de acordo com as recomendações da norma ISO 10272-1:2017. Para a confirmação do gênero e espécie por reação em cadeia da polimerase (PCR), utilizamos os primers 16-rRNA, GlyA e hypO. Para avaliar a sensibilidade antimicrobiana, utilizamos ágar de Müller-Hinton-sangue a 5% com discos de sensibilidade de azitromicina (15 μg) e eritromicina (15 μg). A detecção do gene ermB em cepas com fenótipos resistentes foi feita por PCR convencional. Resultados. Obtivemos um total de 117 amostras positivas (97,5%), das quais 100% foram compatíveis com Campylobacter coli (teste do hipurato negativo) e confirmadas por PCR. Na avaliação da resistência antimicrobiana em placa para azitromicina e eritromicina, 100% das cepas apresentaram fenótipo de resistência a estes macrolídeos, enquanto a PCR para a detecção do gene ermB indicou um total de 62 cepas positivas (53%), que foram confirmadas por sequenciamento. Conclusões. Estes resultados demonstram que as carcaças de frango comercializadas nos mercados de Lima apresentam contaminação por C. coli com alta resistência a macrolídeos, o que pode ser atribuído à presença do gene ermB.

Genetic diversity and zoonotic potential of rotavirus A strains in the southern Andean highlands, Peru.

Interspecies transmission is an important mechanism of evolution and contributes to rotavirus A (RVA) diversity. In order to evaluate the detection frequency, genetic diversity, epidemiological characteristics and zoonotic potential of RVA strains in faecal specimens from humans and animals cohabiting in the same environment in the department of Cusco, Peru, by molecular analysis, 265 faecal specimens were obtained from alpacas, llamas, sheep and shepherd children, and tested for RVA by RT-PCR. Genotyping was performed by multiplex PCR and sequence analysis. Rotavirus A was detected in 20.3% of alpaca, 47.5% of llama, 100% of sheep and 33.3% of human samples. The most common genetic constellations were G3-P[40]-I8-E3-H6 in alpacas, G1/G3-P[8]-I1-E1-H1 in llamas, G1/G3/G35-P[1]/P[8]-I1-E1-H1 in sheep and G3-P[40]-I1/I8-E3-H1 in humans. The newly described genotypes P[40] and P[50] were identified in all host species, including humans. Genotyping showed that the majority of samples presented coinfection with two or more RVA strains. These data demonstrate the great genetic diversity of RVA in animals and humans in Cusco, Peru. Phylogenetic analysis suggested that the strains represent zoonotic transmission among the species studied. Due to the characteristics of the human and animal populations in this study (cohabitation of different host species in conditions of poor sanitation and hygiene), the occurrence of zoonoses is a real possibility.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV) 1-7-4-type strains in Peru.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. While PRRSV has been endemic in North America since 1989, it was not until 1999 that the virus was first described in South America. Notably, recently an increased number of PRRSV outbreaks have been reported in South American countries. However, epidemiological information related to these outbreaks is limited and the genetic characteristics of the PRRSV strains circulating in the region are poorly understood. In this study, we describe the genetic analyses of PRRSV strains associated with severe PRRS outbreaks in Peru. Samples originating from 14 farms located in two Departments in Peru (Lima and Arequipa), were subjected to RT-PCR amplification of the PRRSV ORF5 gene and sequencing followed by restriction fragment length polymorphism (RFLP) analysis. Results demonstrated the circulation of PRRSV-2 in Peru. Notably ORF5 RFLP typing revealed that 15 (75%) of the PRRSV strains detected in this study belong to the RFLP 1-7-4 type. Phylogenetic analysis showed that the Peruvian strains are closely related to the highly virulent PRRSV 1-7-4 strains that emerged in the US in 2013-2014. Results here indicate the presence of highly virulent PRRSV 1-7-4 strains in Peru and provide important information on the geographical distribution of PRRSV, confirming the recent geographical expansion of this important swine pathogen towards South America.

Molecular epidemiology of coronavirus in faeces of Brazilian calves and Peruvian camelid herds.

INTRODUCTION: The enteric disorders represent a serious hazard for bovine and camelid breeding. The aim of this study was to examine the frequency of detection and molecular characteristics of enteric coronavirus (CoV) infections in cattle, alpaca, and llama herds bred in family-based farms in Brazil and Peru. METHODOLOGY: Stool samples were collected from calves from Brazil and camelids from Peru for detection and characterization of CoV by reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. RESULTS: 46.5% (47/101) samples from calves and 26.8% (70/261) from alpaca tested positive for CoV. All strains belong to lineage A1 of the Betacoronavirus genus. Phylogenetic analysis showed high identity between CoV strains detected in calves and alpacas. CONCLUSIONS: This study characterised CoV strains from dairy cattle herds in the state of Rio de Janeiro, Brazil, and indicated that this virus is spread among the state herds. The results also indicate widespread circulation of CoV among the alpacas of Cuzco, Peru.

Identification of two novel Rotavirus A genotypes, G35 and P[50], from Peruvian alpaca faeces.

Rotavirus A (RVA) Alp11B was detected from a neonatal Peruvian alpaca presenting with diarrhea, and the Alp11B VP7, VP4, VP6, NSP4, and NSP5 genes were sequenced. The partial genotype constellation of this strain, RVA/Alpaca-wt/PER/Alp11B/2010, was determined to be G35-P[50]-I13-E16-H6.

Whole-genome characterization of a Peruvian alpaca rotavirus isolate expressing a novel VP4 genotype.

The SA44 isolate of Rotavirus A (RVA) was identified from a neonatal Peruvian alpaca presenting with diarrhea, and the full-length genome sequence of the isolate (designated RVA/Alpaca-tc/PER/SA44/2014/G3P[40]) was determined. Phylogenetic analyses showed that the isolate possessed the genotype constellation G3-P[40]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which differs considerably from those of RVA strains isolated from other species of the order Artiodactyla. Overall, the genetic constellation of the SA44 strain was quite similar to those of RVA strains isolated from a bat in Asia (MSLH14 and MYAS33). Nonetheless, phylogenetic analyses of each genome segment identified a distinct combination of genes. Several sequences were closely related to corresponding gene sequences in RVA strains from other species, including human (VP1, VP2, NSP1, and NSP2), simian (VP3 and NSP5), bat (VP6 and NSP4), and equine (NSP3). The VP7 gene sequence was closely related to RVA strains from a Peruvian alpaca (K'ayra/3368-10; 99.0% nucleotide and 99.7% amino acid identity) and from humans (RCH272; 95% nucleotide and 99.0% amino acid identity). The nucleotide sequence of the VP4 gene was distantly related to other VP4 sequences and was designated as the reference strain for the new P[40] genotype. This unique genetic makeup suggests that the SA44 strain emerged from multiple reassortment events between bat-, equine-, and human-like RVA strains.

Outbreak of diarrhea among preweaning alpacas (Vicugna pacos) in the southern Peruvian highland.

INTRODUCTION: Infections, particularly diarrheal infections, are a major cause of neonatal death in South American camelids. The aim of this study was to identify the pathogens that could have caused the recent diarrhea outbreak among the alpacas in Silli, Cusco, located in the southern Peruvian highland. METHODOLOGY: Spleen, kidney, and intestine tissue along with fecal and intestinal lavage samples were obtained from 50 one- to five-week-old alpacas and analyzed for the presence of parasites, bacteria, and viruses. RESULTS: Laboratory testing of the 50 crias included in this study revealed that 80% were infected with Eimeria spp., 40% with coronavirus, 34% with E. coli, 32% with rotavirus, 22% with Clostridium spp., and 20% with Cryptosporidium spp. Of these 50 alpaca crias, 20 presented with a single infection (19 positive for Eimeria spp. and 1 positive for rotavirus). Co-infections with up to four pathogens occurred in 60% of the samples. The significance of such infections is not clear, but it is noteworthy that the animals suffering from necrotic and/or hemorrhagic enteritis presented with quadruple infections. It is likely that co-infections increase the severity of the disease. CONCLUSIONS: These data show that multiple pathogens circulate among young alpaca crias and could be associated with diarrheal disease in these animals. The findings from this study warrant the provision of subsidies for future assessment of the potential economic impact of these infections on the productivity of the Peruvian alpaca industry.