Evolution and divergence of the genetic lineage Desmodus rotundus/Artibeus lituratus of rabies virus in São Paulo State.

The last record of a rabies case caused by the dog-specific rabies virus (RABV) lineage in dogs or cats in São Paulo State was in 1998. From 2002 to 2021, 57 cases of rabies in these animals were reported, and the vast majority (51) were genetically characterized as belonging to the Desmodus rotundus/Artibeus lituratus RABV lineage. However, it is not currently possible to infer which of these bats is the source of infection by genome sequencing of RABV isolates. The aims of this study were (a) to characterize the Desmodus rotundus/Artibeus lituratus lineage to determine the relationships between the RABV lineages and each reservoir, (b) to assess the phylogeny and common ancestors of the RABV lineages found in D. rotundus and A. lituratus, and (c) to further understand the epidemiology and control of rabies. In this study, we genetically analyzed 70 RABV isolates from São Paulo State that were received by the Virology Laboratory of the Pasteur Institute of São Paulo between 2006 and 2015. Of these isolates, 33 were associated with the hematophagous bat D. rotundus and 37 with the fruit bat A. lituratus. A genomic approach using phylogenetic analysis and nucleotide sequence comparisons demonstrated that these isolates belonged to the same genetic lineage of RABV. We also found that, in São Paulo State, the D. rotundus/A. lituratus lineage could be subdivided into at least four phylogenetic sublineages: two associated with D. rotundus and two with A. lituratus. These results are of importance for the epidemiological surveillance of rabies in São Paulo.

Emergence of Avian coronavirus Escape Mutants Under Suboptimal Antibody Titers.

To perform a quasispecies assessment of the effect of vaccine combinations and antibody titers on the emergence of Avian coronavirus (AvCoV) escape mutants, 5-week-old males from a commercial chicken breeder lineage were vaccinated intramuscularly with one dose of a monovalent (genotype GI-1) or a bivalent (genotypes GI-1 and GI-11 (n = 40 birds/group) AvCoV vaccine. Seven birds were kept as controls. Six weeks later, pools of sera of each group were prepared and incubated at virus neutralization doses of 10 and 10-1 with the Beaudette strain (GI-1) of AvCoV in VERO cells. Rescued viruses were then submitted to genome-wide deep sequencing for subconsensus variant detection. After treatment with serum from birds vaccinated with the bivalent vaccine at a titer of 10-1, an F307I variant was detected in the spike glycoprotein that mapped to an important neutralizing region, which indicated an escape mutant derived from natural selection. Further variants were detected in nonstructural proteins and non-coding regions that are not targets of neutralizing antibodies and might be indicators of genetic drift. These results indicate that the evolution of AvCoV escape mutants after vaccination depends on the type of vaccine strain and the antibody titer and must be assessed based on quasispecies rather than consensus dominant sequences only because quasispecies may be otherwise undetected.

Comparison among three different serological methods for the detection of equine influenza virus infection.

The equine influenza virus (EIV) H3N8 subtype is responsible for all EIV outbreaks worldwide while the H7N7 subtype is less pathogenic and is considered extinct as it has not been confirmed in outbreaks since 1980. Although EIV is enzootic in Brazil, few reports describe the actual EIV antibody status in the country. The aims of this study were: - to evaluate the efficiency of different serum treatments described by the World Organisation for Animal Health (OIE) and the World Health Organization (WHO) to remove non-specific haemagglutination inhibitors for the haemagglutination inhibition (HI) assay for EIV - to evaluate the presence of EIV antibodies by HI, enzyme-linked immunosorbent assay and agar gel immunodiffusion in 83 non-vaccinated equines from São Paulo State - to evaluate a strategy to better analyse equine sera for EIV antibodies. Although there was no statistical difference among treatments, receptor-destroying enzyme treatment followed by chicken erythrocyte adsorption showed more consistent results, which corroborate the OIE and WHO recommendation to use this treatment preferentially. The HI results suggest equine H3N8 virus circulation among the animals tested from São Paulo State. The algorithm suggested here could be used to guide antibody detection against equine influenza virus in equines, improving the test specificity by aiming to avoid false positive results.

Tous les foyers de grippe équine dans le monde sont dus au sous-type H3N8 du virus. Le sous-type H7N7, moins pathogène, est considéré comme éteint, sa présence n'ayant été confirmée dans aucun des foyers enregistrés depuis 1980. Au Brésil, la grippe équine est enzootique mais la prévalence d'anticorps dans le pays est peu documentée. La présente étude avait trois objectifs : ­ évaluer l'efficacité de plusieurs traitements de sérums décrits par l'Organisation mondiale de la santé animale (OIE) et l'Organisation mondiale de la santé (OMS) sur la suppression des inhibiteurs d'hémagglutination non spécifiques, afin de pouvoir utiliser l'épreuve d'inhibition de l'hémagglutination pour la détection de la grippe équine, ­ évaluer la présence d'anticorps dirigés contre la grippe équine chez 83 chevaux non vaccinés de l'état de São Paulo en utilisant l'inhibition de l'hémagglutination, l'épreuve immuno-enzymatique (ELISA) et l'épreuve d'immunodiffusion en gélose (IDG) ; ­ évaluer une stratégie visant à améliorer les techniques sérologiques de détection des anticorps dirigés contre la grippe équine. S'il n'y a pas eu de différence statistique significative entre les traitements, celui faisant appel à l'enzyme de destruction du récepteur suivi d'une adsorption sur érythrocytes de poule a permis d'obtenir les résultats les plus cohérents, ce qui corrobore les recommandations de l'OIE et de l'OMS en faveur de ce traitement. Les résultats obtenus au moyen de l'inhibition de l'hémagglutination indiquent que le virus H3N8 est présent parmi les animaux testés de l'état de São Paulo. L'algorithme présenté par les auteurs pourrait servir de modèle pour détecter la présence d'anticorps dirigés contre le virus de la grippe équine chez les chevaux : en effet, il permet d'éviter les résultats faussement positifs, ce qui améliore la spécificité du test utilisé.

El subtipo H3N8 del virus de la gripe equina (VGE) es el agente etiológico de todos los brotes que se producen en el mundo, mientras que el subtipo H7N7, menos patogénico, se da por extinto, en la medida en que desde 1980 no se ha confirmado su intervención en brote alguno. Aunque en el Brasil el VGE es enzoótico, existen pocos trabajos que den cuenta de la situación real del país en cuanto a la presencia de anticuerpos contra el virus. Los autores describen un estudio que perseguía los siguientes objetivos: ­ evaluar la eficacia de distintos tratamientos séricos descritos por la Organización Mundial de Sanidad Animal (OIE) y la Organización Mundial de la Salud (OMS) para eliminar los inhibidores inespecíficos de la hemaglutinación con objeto de aplicar la técnica de inhibición de la hemaglutinación a la detección del VGE; ­ evaluar la presencia de anticuerpos contra el VGE por inhibición de la hemaglutinación, ensayo inmunoenzimático (ELISA) e inmunodifusión en gel de agar en 83 ejemplares equinos no vacunados del estado de São Paulo; ­ evaluar una estrategia encaminada a analizar más eficazmente sueros equinos para detectar en ellos anticuerpos anti-VGE. Aunque no se observaron diferencias estadísticamente significativas entre los tratamientos, el uso de enzimas destructores de receptores seguido de la técnica de adsorción de eritrocitos de pollo arrojó resultados más coherentes, cosa que avala la recomendación de la OIE y la OMS de privilegiar este tratamiento. Los resultados obtenidos por inhibición de la hemaglutinación parecen indicar que el virus H3N8 equino circula entre los animales analizados del estado de São Paulo. El algoritmo aquí propuesto podría servir de guía para detectar en equinos la presencia de anticuerpos contra el VGE. Puesto que apunta a evitar falsos positivos, su aplicación mejoraría la especificidad de la prueba.

Chlamydia felis: Lack of association between clinical signs and the presence of the cryptic plasmid.

Chlamydia felis is an obligate intracellular bacterial pathogen that infects cats, causing severe conjunctivitis associated with upper respiratory tract disease (URTD). In the present study, 186 cats from three non-commercial catteries in São Paulo, SP, Brazil were evaluated. The detection of Chlamydia felis was performed by PCR. The clinical severity was scored from 1 to 4, with a score of 4 as the most severe manifestation. The total occurrence of C. felis was of 18.82% (35/186) of cats overall, but notably, 58.06% (18/31) of infected cats originated from a single cattery. All animals harboring C. felis had URTD clinical signs and higher scores (3 and 4). In addition, C. felis occurrence was associated with the presence of cryptic plasmid. However, the virulence and clinical severity were not correlated.

Betacoronavirus 1 in alpacas (Vicugna pacos) in the High Peruvian Andes.

Genetic sequences highly related to Bovine coronavirus (BCoV) were detected in fecal samples from Peruvian 1-3 week old alpaca crias located on six farms in Puno department, some of which shared pastures with cattle. A total of 60 samples were screened for coronavirus using a nested PCR amplification of a fragment of the RNA-dependent RNA polymerase (RdRp) gene. Sequences from 11 positive samples were highly similar to the Kakegawa, Quebec and Mebus BCoV strains (99.5-100.0%) and 99.2% identical to an alpaca Coronavirus (CoV) previously detected in the USA. The detection of genetic sequences related to BCoV from Peruvian alpaca crias suggests possible role of this virus on enteric disorders etiology in the High Andes.

Occurrence and characterization of rotavirus A in broilers, layers, and broiler breeders from Brazilian poultry farms.

Rotaviruses are a major cause of diarrhea in humans and animals, including several mammalian and avian species. Using different PCR protocols, we report the occurrence of rotavirus A in 21 (53.84%; 21/39) from 39 fecal pool samples of broilers, layers, and broiler breeders from Brazilian avian farms. We typed the G5, G8, G11, G19, and P[31] genotypes.

An Avian coronavirus in quail with respiratory and reproductive signs.

An Avian coronavirus was detected in pools of lungs, tracheas, female reproductive tracts, kidneys, and enteric contents from quail (Coturnix coturnix japonica) and laying hen flocks, with and without infectious bronchitis (IB)-like signs, cohoused in farms located in two states of southeastern Brazil during 2009-2010. Although Avian metapneumovirus subtype B was found in two layers samples, Newcastle disease virus was not found in quail or in hens. Based on DNA sequences for the 3'-untranslated region and the gene encoding the RNA-dependent RNA polymerase, this avian coronaviruses in quail is an IB virus-like gammacoronavirus.

Detection of Genetic characterization of Porcine circovirus 2 (PCV2) in Brazilian wildlife boars.

A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.

Bovine rotavirus in turkeys with enteritis.

Rotaviruses are the main agents responsible for diarrhea in different animal species and for infantile gastroenteritis. These viruses have been isolated from various avian species and have often been associated with poult enteritis and mortality syndrome. Nevertheless, the knowledge of rotavirus infection in turkeys is scarce. Six group A rotavirus strains obtained from pooled enteric contents of diarrheic turkeys were isolated in MA-104 cell culture and typed as G(6)P(1), a typical bovine rotavirus genotype. Additionally, the electropherotypes showed a migration pattern identical to the Nebraska calf diarrhea virus, and the complete NSP4 gene phylogeny showed that all six strains segregated in the genotype E2. Taken together, these results point toward a cattle-to-turkey rotavirus transmission. As a conclusion, bovine-origin rotavirus can be found in turkeys, and this transmission route must now be considered for the improvement of the health status in turkey farms.

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil.

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G2254/D752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A2254/N752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Molecular epidemiology of avian infectious bronchitis in Brazil from 2007 to 2008 in breeders, broilers, and layers.

Multiple lineages of Brazilian strains from 2007 to 2008 of avian infectious bronchitis virus (IBV) were detected in flocks of breeders, broilers, and layers. Organs samples from 20 IBV-positive flocks with variable clinical signs were submitted to the partial amplification of S gene (nucleotides 726-1071) of IBV. Fifteen of the 20 sequenced strains segregated in a unique Brazilian cluster subdivided in three subclusters (Brazil 01, 02, and 03). Whereas three strains could be classified as Massachusetts (Mass) genotype, the remaining two strains, originating from flocks with reproductive and respiratory disorders, grouped within the 4/91-793B genotype, a genotype that has not been detected before in Brazil. The potential relevance of the findings to the poultry industry is discussed because the low level of identity of the sequenced part of the S gene from 17 of 20 detected field strains and the vaccines of the Massachusetts serotype used suggest that the level of cross-protection by the Massachusetts vaccines might be low.

Molecular diversity of Alphacoronavirus 1 in dogs and cats in Colombia.

Alphacoronavirus 1 (subgenus Tegacovirus, genus Alphacoronavirus, family Coronaviridae), which encompasses transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV) and canine coronavirus (CCoV), is an important pathogen that can cause severe gastroenteritis and is distributed worldwide. CCoV has two different genotypes: CCoV type I, which has a high identity with FCoV-I, and CCoV type II, which is divided into two subtypes, CCoV IIa (pantropic) and CCoV IIb, which is related to FCoV-II and has been involved in multiple recombination events. Between 2014 and 2018, 43 fecal samples from puppies and young dogs under 1 year of age with hemorrhagic enteritis and from 5 cats under 2 years of age with ascites or thoracic effusion were collected by a private veterinary practice in Bogotá, Colombia. A screening for Coronavirus via RT-PCR (nsp12) and PCR amplification of Canine protoparvovirus (VP1) revealed 27.1% (13/49) and 72.9% (35/49) positive samples, respectively. Positive samples for coronavirus were tested for M, N, S and the sequences grouped in the FCoV, CCoV-I and CCoV-IIb clusters that were distant from the pantropic type (IIa). The N gene formed two clusters, one exclusively with samples from this study in subtype II and another with strains in subtype I. For gene S (subtype I), the samples clustered with the Brazilian samples, while samples positive for S subtype IIb grouped into a cluster distinct from the other reference sequences. The prevalence of coronaviruses identified in this study is within the range reported by different countries worldwide.

Simultaneous presence of bovine papillomavirus in blood and in short-term lymphocyte cultures from dairy cattle in Pernambuco, Brazil.

Bovine papillomaviruses (BPV) are the causal agents of benign and malignant lesions; they can cause dramatic economic losses in cattle. Although 10 virus types have been described, three types are most common in tumors, namely BPV-1, -2 and -4. Previous studies have reported BPV in blood cells and the possibility of blood acting as a latent virus site and/or transmission agent of virus dissemination. We studied a Holstein dairy herd in Pernambuco, Brazil, in which several animals showed severe cutaneous papillomatosis, without previous determination of BPV types. Blood samples and short-term lymphocyte cultures were collected from 54 cows. We compared the BPV types detected in peripheral blood to those identified in the respective lymphocyte cultures: BPV-1 was detected in 74% and BPV-2 in 87% of the whole blood samples. Simultaneous virus presence (BPV-1 and BPV-2) was found in 65% of the blood samples. BPV-1 or BPV-2 were detected in the lymphocyte cultures in 93% of the samples, and both in 89%. The detection of viral DNA in whole blood and in lymphocyte cultures is evidence that this virus is carried by lymphocytes.

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction.

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.

Can established cultured papilloma cells harbor bovine papillomavirus?

Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

Molecular epidemiology identifies only a single rabies virus variant circulating in complex carnivore communities of the Serengeti.

Understanding the transmission dynamics of generalist pathogens that infect multiple host species is essential for their effective control. Only by identifying those host populations that are critical to the permanent maintenance of the pathogen, as opposed to populations in which outbreaks are the result of 'spillover' infections, can control measures be appropriately directed. Rabies virus is capable of infecting a wide range of host species, but in many ecosystems, particular variants circulate among only a limited range of potential host populations. The Serengeti ecosystem (in northwestern Tanzania) supports a complex community of wild carnivores that are threatened by generalist pathogens that also circulate in domestic dog populations surrounding the park boundaries. While the combined assemblage of host species appears capable of permanently maintaining rabies in the ecosystem, little is known about the patterns of circulation within and between these host populations. Here we use molecular phylogenetics to test whether distinct virus-host associations occur in this species-rich carnivore community. Our analysis identifies a single major variant belonging to the group of southern Africa canid-associated viruses (Africa 1b) to be circulating within this ecosystem, and no evidence for species-specific grouping. A statistical parsimony analysis of nucleoprotein and glycoprotein gene sequence data is consistent with both within- and between-species transmission events. While likely differential sampling effort between host species precludes a definitive inference, the results are most consistent with dogs comprising the reservoir of rabies and emphasize the importance of applying control efforts in dog populations.

Orchitis in roosters with reduced fertility associated with avian infectious bronchitis virus and avian metapneumovirus infections.

The pathogenesis of infection involving both infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) causes reproductive damage in hens after viral replication in the epithelium of the oviduct, resulting in loss of cilia and degeneration and necrosis of the epithelial and glandular cells. Although IBV has been indicated as a possible cause of the formation of calcium stones in the epididymus of roosters, a definitive association has not been confirmed. This report describes the detection of IBV and aMPV in the testes of roosters from a Brazilian poultry broiler breeder's flock with epididymal stones and low fertility. Samples of testis, trachea, and lungs from breeder males aged 57 wk were positive for IBV by reverse transcriptase-polymerase chain reaction (RT-PCR), and virus isolation and testis samples were also positive for aMPV by RT-PCR. The inoculation of testis samples into embryonated chicken eggs via the allantoic cavity resulted in curled, hemorrhagic, and stunted embryos typical of IBV infection. The allantoic fluid was positive by RT-PCR aimed to amplify the region coding for the S1 subunit of the IBV S gene, but it was not positive for aMPV. Sequence analysis of the amplified fragment revealed a close relationship with European IBV genotype D274, previously unreported in Brazil. These results indicate that IBV and perhaps aMPV are likely to have played a role in the pathogenesis of the testicular disease described and should be regarded as factors that can influence male fertility disease in chickens.

Molecular characterization of infectious bronchitis virus strains isolated from the enteric contents of Brazilian laying hens and broilers.

Infectious bronchitis virus (IBV) is the causative agent of avian infectious bronchitis, which is characterized by respiratory, reproductive, and renal signs. However, the role of IBV as an enteric pathogen in still controversial. In Brazil, antigenic groups of IBV divergent from the Massachusetts serotype used for vaccination schedules in that country have already been demonstrated. The present study aimed to assess the different genotypes of IBV in Brazilian commercial poultry flocks by partial sequencing of the S1 amino-terminus coding region using enteric contents as samples and examine their relationship with the vaccine serotype currently in use. Samples of enteric contents were taken as pools of five birds from each of 18 poultry farms (17 broiler and one laying farm) from five Brazilian states between 2002 and 2006. Birds were presenting watery diarrhea and poor general condition but were without respiratory, renal, or reproductive signs. Conventional antibacterial and anticoccidial therapies were unsuccessful and, furthermore, all samples proved negative for rotavirus, reovirus, and astrovirus. Eleven IBV samples were isolated in embryonated eggs and resulted in S1 sequences. Phylogenetic analysis showed that these segregated into an exclusive cluster, close to serotype D274, but distant from Massachusetts. Mean amino acid identity amongst these Brazilian strains was 94.07%; amongst these and serotypes D274, 4/91, and Massachusetts, mean amino acid identity was 77.17%, 69.94%, and 68.93%, respectively. In conclusion, the presence of genotype variant strains of IBV in Brazilian poultry flocks has been demonstrated and might be the reason for the unsuccessful control of IBV in Brazil. Furthermore, these results also strengthen the implications of IBV in enteric diseases of poultry.

Gamma and Deltacoronaviruses in quail and pheasants from Northern Italy1.

In view of the restricted knowledge on the diversity of coronaviruses in poultry other than chicken, this study aimed to investigate the genetic diversity of coronaviruses in quail, pheasant, and partridge from two regions of Northern Italy. To this end, pools of tracheal and cloacal swabs from European quail (Coturnix Coturnix) and intestinal tract from pheasants (Phasianus Colchicus) and partridge (Perdix Perdix) flocks, with or without enteric signs, were collected during 2015. Avian coronavirus (Gammacoronavirus) was detected in quail not vaccinated against Infectious Bronchitis Virus (IBV) and in pheasants vaccinated with an IBV Massachusetts serotype. Based on DNA sequences for the gene encoding the S protein, the avian coronaviruses detected in the quail and pheasant are related to the IBV 793B and Massachusetts types, respectively. However, RNA-dependent RNA polymerase (RdRp) analyses showed the susceptibility of quail also to Deltacoronaviruses, suggesting that quail and pheasant avian coronaviruses share spike genes identical to chicken IBV spike genes and quail might host Deltacoronavirus.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a-c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.