Unraveling swine hepatitis E in the central region of Argentina through ELISA development and epidemiological insights.

Hepatitis E virus (HEV) is a public health concern globally, causing acute viral hepatitis in humans. Genotype-3 HEV (HEV-3), the most frequently genotype detected in South America, is zoonotic and the main reservoirs are the domestic pig and wild boar. Circulation of HEV-3 in Argentina has been confirmed in humans as well as in pig herds, wild boar and environmental waters. However, data are scarce mainly due to the inaccessibility of serological assays in this country. In order to provide insights in the epidemiology of HEV in swine in Argentina, we developed an indirect ELISA based on the native recombinant protein ORF2 and conducted a serological survey to determine the prevalence of seropositive swine in small-scale pig farms in the central region of Argentina. The method was evaluated in a panel of 157 serum samples, resulting in relative sensitivity of 98.6 % (95 % CI 95 %-100 %) and relative specificity of 97.7 % (95 % CI 94 %-100 %) compared to a commercial test. An almost perfect agreement was obtained between the two tests (Kappa index of 0.961). A survey on 294 samples from 49 small-scale farms resulted in a seropositivity rate of 54 %. Seropositive animals were found in 34 out of 49 (69.4 %) farms. Most of the farms (70.6 %) had over 50 % of seropositive animals. The wide spreading of HEV in the swine population of Tandil, Argentina, underscore the need to better understand the epidemiology of HEV in the region, enabling the implementation of targeted interventions to mitigate the impact of this virus on public health.

The Prevalence and Genetic Diversity of PCV3 and PCV2 in Colombia and PCV4 Survey during 2015-2016 and 2018-2019.

Four genotypes of circovirus have been recognized in swine, with PCV2 and PCV3 being the most associated with clinical manifestations, while PCV4 does not have a defined disease. In addition, PCV2 is associated with different syndromes grouped as diseases associated with porcine circovirus (PCVAD), while PCV3 causes systemic and reproductive diseases. In the present study, we retrospectively detected PCV2, PCV3, and PCV4 in Colombia during two periods: A (2015-2016) and B (2018-2019). During period A, we evaluated stool pools from the 32 Colombian provinces, finding a higher prevalence of PCV3 compared to PCV2 as well as PCV2/PCV3 co-infection. Furthermore, we determined that PCV3 had been circulating since 2015 in Colombia. Regarding period B, we evaluated sera pools and tissues from abortions and stillborn piglets from the five provinces with the highest pig production. The highest prevalence found was for PCV3 in tissues followed by sera pools, while PCV2 was lower and only in sera pools. In addition, PCV2/PCV3 co-infection in sera pools was also found for this period. The complete genome sequences of PCV3 and PCV3-ORF2 placed the Colombian isolates within clade 1 as the majority in the world. For PCV2, the predominant genotype currently in Colombia is PCV2d. Likewise, in some PCV3-ORF2 sequences, a mutation (A24V) was found at the level of the Cap protein, which could be involved in PCV3 immunogenic recognition. Regarding PCV4, retrospective surveillance showed that there is no evidence of the presence of this virus in Colombia.

Oral Vaccination with Hepatitis E Virus Capsid Protein and Immunobiotic Bacterium-Like Particles Induce Intestinal and Systemic Immunity in Mice.

The hepatitis E virus (HEV) genotype 3 (GT3) is an emergent pathogen in industrialized countries. It is transmitted zoonotically and may lead to chronic hepatitis in immunocompromised individuals. We evaluated if the major antigen of HEV, the capsid protein, can be used in combination with immunobiotic bacterium-like particles (IBLP) for oral vaccination in a mouse model. We have cloned and expressed the RGS-His5-tagged HEV GT3 capsid protein (ORF2) in E. coli and purified it by NiNTA. IBLP were obtained from two immunobiotic Lactobacillus rhamnosus strains acid- and heat-treated. ORF2 and the IBLP were orally administered to Balb/c mice. After three oral immunizations (14-day intervals), blood, intestinal fluid, Peyer´s patches, and spleen samples were drawn. IgA- and IgG-specific antibodies were determined by ELISA. Mononuclear cell populations from Peyer's patches and spleen were analyzed by flow cytometry, and the cytokine profiles were determined by ELISA to study cellular immunity. Orally administered recombinant ORF2 and IBLP from two L. rhamnosus strains (CRL1505 and IBL027) induced both antigen-specific humoral and cellular immune responses in mice. IBLP027 was more effective in inducing specific secretory IgA in the gut. IFN-γ, TNF-α, and IL-4 were produced by Peyer's plaques lymphocytes stimulated with ORF2 ex vivo suggesting a mixed Th1/Th2-type adaptive immune response in immunized mice. Oral vaccines are not invasive, do not need to be administered by specialized personal, and elicit both systemic and local immune responses at the port of entry. Here, we present an experimental oral vaccine for HEV GT3, which could be further developed for human and/or veterinary use.

Molecular characterization of the ORF2 of Torque teno sus virus 1a and Torque teno sus virus 1b detected in cases of postweaning multisystemic wasting syndrome in Mexico.

Worldwide Torque teno sus virus (TTSuV, genus Iotatorquevirus) species have been regarded as possible agents associated with porcine circovirus-associated disease. Iotatorquevirus species possess high genomic variability, suggesting that diverse genotypes are widely geographically distributed. In this study, we validated the genomic variability of Iotaroquevirus species in pigs with postweaned multisystemic wasting syndrome. Genomic DNA from nine TTSuV1a-positive tissues and 15 TTSuV1b-positive tissues was used to amplify the complete ORF2 of each species by nested PCR to perform a molecular characterization. It was found that Mexican TTSuV1a sequences belong to genotype B, sharing phylogenetic origin, high nucleic acid and amino acid sequence similarity and dominant epitope conformation with commercially linked countries, such as the United States, Canada and China, whereas the Mexican TTSuV1b sequences belong to genotype A, being more divergent among each other and displaying low nucleotide identity with worldwide genotype A sequences. In both Iotatorquevirus species, a PTPase-like signature motif was identified in the predicted amino acid sequence, being more conserved for Mexican TTSuV1b sequences than for Mexican TTSuV1a sequences, in which several substitutions were observed. These changes may influence the conformation of dominant epitopes as different arrays were determined among TTSuV1a genotypes. ORF2 variability may account for pathogenic differences by modifying viral replication and immune response, as depicted for human TTV.

Primer design for complete sequencing of the Avastrovirus ORF2 gene / Desenho de primers para o sequenciamento completo do gene ORF2 em Avastrovirus

Avastrovirus infection is associated with enteric disease, nephritis, and hepatitis in birds. In this study, we present a protocol for the complete sequencing of the ORF2 gene of avian nephritis virus (ANV), chicken astrovirus (CAstV), and turkey astrovirus type 1 (TAstV-1) using a conventional Sanger technique. Previously and newly designed primer pairs targeting both the conserved flanking and internal regions of the ORF2 gene of these three viruses were used. The information derived from the astroviral sequences obtained in this study is fundamental for characterizing this virus and providing data regarding several aspects of disease epidemiology and prevention.(AU)

As infecções por avastrovírus estão associados à doença entérica, nefrite e hepatite em aves. Aqui, nos presentamos um protocolo planejado para o sequenciamento completo do gene ORF2 em Avian Nephritis Vírus (ANV), Chicken Astrovirus (CAstV) e Turkey Astrovirus tipo 1 (TAstV-1), usando a técnica de sequenciamento convencional de Sanger. Foram usados primers previamente descritos e desenhados neste estudo, tendo como alvo as regiões conservadas flanqueadoras e internas dentro do gene ORF2 nos três vírus. O conhecimento destas sequencias é um elemento chave para caracterizar o vírus e prover de dados em diversos aspectos da epidemiologia e prevenção da doença.(AU)

Primer design for complete sequencing of the Avastrovirus ORF2 gene / Desenho de primers para o sequenciamento completo do gene ORF2 em Avastrovirus

Avastrovirus infection is associated with enteric disease, nephritis, and hepatitis in birds. In this study, we present a protocol for the complete sequencing of the ORF2 gene of avian nephritis virus (ANV), chicken astrovirus (CAstV), and turkey astrovirus type 1 (TAstV-1) using a conventional Sanger technique. Previously and newly designed primer pairs targeting both the conserved flanking and internal regions of the ORF2 gene of these three viruses were used. The information derived from the astroviral sequences obtained in this study is fundamental for characterizing this virus and providing data regarding several aspects of disease epidemiology and prevention.

As infecções por avastrovírus estão associados à doença entérica, nefrite e hepatite em aves. Aqui, nos presentamos um protocolo planejado para o sequenciamento completo do gene ORF2 em Avian Nephritis Vírus (ANV), Chicken Astrovirus (CAstV) e Turkey Astrovirus tipo 1 (TAstV-1), usando a técnica de sequenciamento convencional de Sanger. Foram usados primers previamente descritos e desenhados neste estudo, tendo como alvo as regiões conservadas flanqueadoras e internas dentro do gene ORF2 nos três vírus. O conhecimento destas sequencias é um elemento chave para caracterizar o vírus e prover de dados em diversos aspectos da epidemiologia e prevenção da doença.

Turkey Astrovirus Type 1 (TAstV-1) and Chicken Astrovirus (CAstV) Detection in Brazilian Chicken Flocks.

Astrovirus is a common cause of enteritis in humans and domestic animals. Here we report the detection of turkey astrovirus type 1 (TAstV-1) and chicken astrovirus (CAstV) in avian farms. Sixty fecal sample pools (five or six birds of the same flock), from chickens without apparent clinical symptoms of enteric disease from farms located in six Brazilian states, were screened by an ORF1b PCR, followed by nucleotide sequencing of amplified products and phylogenetic analysis. Six samples tested positive for TAstV-1 and two for CAstV. One positive sample of each detected virus (TAstV-1 and CAstV) had the complete ORF2 sequenced. Data for the ORF2 sequence indicate that Brazilian TAstV-1 was divergent from TAstV-1 (United States), previously described infecting turkeys, and Brazilian CAstV clustered together with the U.K. group, subgroup B-II, associated with enteritis and growth retardation in chicks. This study provides updated information about CAstV and the first report of detection of TAstV-1 in Brazilian chickens, supporting the diagnostic of enteritis and epidemiologic surveillance in poultry health.

Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis.

Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.

Effects of L1-ORF2 fragments on green fluorescent protein gene expression.

The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2) induces premature termination of transcription that is consistent with the effect of ORF2.

Effects of L1-ORF2 fragments on green fluorescent protein gene expression

The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2) induces premature termination of transcription that is consistent with the effect of ORF2.