Identification of virulence factors and antibiotic resistance in Salmonella enterica subsp. enterica serovar Javiana (FARPER-220) isolated from broiler chickens.

Genome sequencing of highly virulent Salmonella enterica subsp. enterica serovar Javiana strain FARPER-220 (ST-1674) isolated from broiler chickens in Peru revealed multiple virulence factors, antibiotic resistance genes, and invasion-related subcategories. The results provide insights into the potential importance of this strain in causing infections in various animals.

Identification and evaluation in-vitro of conserved peptides with high affinity to MHC-I as potential protective epitopes for Newcastle disease virus vaccines.

BACKGROUND: Newcastle disease (ND) is a major threat to the poultry industry, leading to significant economic losses. The current ND vaccines, usually based on active or attenuated strains, are only partially effective and can cause adverse effects post-vaccination. Therefore, the development of safer and more efficient vaccines is necessary. Epitopes represent the antigenic portion of the pathogen and their identification and use for immunization could lead to safer and more effective vaccines. However, the prediction of protective epitopes for a pathogen is a major challenge, especially taking into account the immune system of the target species. RESULTS: In this study, we utilized an artificial intelligence algorithm to predict ND virus (NDV) peptides that exhibit high affinity to the chicken MHC-I complex. We selected the peptides that are conserved across different NDV genotypes and absent in the chicken proteome. From the filtered peptides, we synthesized the five peptides with the highest affinities for the L, HN, and F proteins of NDV. We evaluated these peptides in-vitro for their ability to elicit cell-mediated immunity, which was measured by the lymphocyte proliferation in spleen cells of chickens previously immunized with NDV. CONCLUSIONS: Our study identified five peptides with high affinity to MHC-I that have the potential to serve as protective epitopes and could be utilized for the development of multi-epitope NDV vaccines. This approach can provide a safer and more efficient method for NDV immunization.

Detection and Genomic Characterization of an Avian Influenza Virus Subtype H5N1 (Clade 2.3.4.4b) Strain Isolated from a Pelican in Peru.

Surveillance helps us identify and monitor strains with zoonotic potential. A tracheal swab from a pelican on a Peruvian beach was H5N1 positive (clade 2.3.4.4b) using Oxford Nanopore's MinION platform. The near-complete genome sequence of strain VFAR-140 will aid us in understanding avian influenza epidemiology and spread.

Draft Genome Sequence of an Isolate of Genotype VII Newcastle Disease Virus Isolated from an Outbreak in Fighting Cock in Peru.

This study presents a draft genome sequence of a Newcastle disease virus (NDV) strain (VFAR-136) isolated from a fighting cock (Gallus gallus) in the south of Peru. Strain VFAR-136 is a new report of NDV genotype VII circulating in Peru.

Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models.

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus lead to the generation of new alternatives to improve the current sub-unit vaccines in development. In the present report, the immunogenicity of the Spike RBD of SARS-CoV-2 formulated with an oil-in-water emulsion and a water-in-oil emulsion with squalene was evaluated in mice and hamsters. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays, although the cellular immune response elicited by both adjuvants were similar, the formulation based in water-in-oil emulsion and squalene generated an earlier humoral response as determined by ELISA. Similarly, this formulation was able to stimulate neutralizing antibodies in hamsters. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results have shown the potential of this formulation vaccine to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection.

Preclinical Assessment of IgY Antibodies Against Recombinant SARS-CoV-2 RBD Protein for Prophylaxis and Post-Infection Treatment of COVID-19.

Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Comprehensive virtual screening of 4.8 k flavonoids reveals novel insights into allosteric inhibition of SARS-CoV-2 MPRO.

SARS-CoV-2 main protease is a common target for inhibition assays due to its high conservation among coronaviruses. Since flavonoids show antiviral activity, several in silico works have proposed them as potential SARS-CoV-2 main protease inhibitors. Nonetheless, there is reason to doubt certain results given the lack of consideration for flavonoid promiscuity or main protease plasticity, usage of short library sizes, absence of control molecules and/or the limitation of the methodology to a single target site. Here, we report a virtual screening study where dorsilurin E, euchrenone a11, sanggenol O and CHEMBL2171598 are proposed to inhibit main protease through different pathways. Remarkably, novel structural mechanisms were observed after sanggenol O and CHEMBL2171598 bound to experimentally proven allosteric sites. The former drastically affected the active site, while the latter triggered a hinge movement which has been previously reported for an inactive SARS-CoV main protease mutant. The use of a curated database of 4.8 k flavonoids, combining two well-known docking software (AutoDock Vina and AutoDock4.2), molecular dynamics and MMPBSA, guaranteed an adequate analysis and robust interpretation. These criteria can be considered for future screening campaigns against SARS-CoV-2 main protease.

A transient increase in MHC-IIlow monocytes after experimental infection with Avibacterium paragallinarum (serovar B-1) in SPF chickens.

Infectious coryza (IC), an upper respiratory tract disease affecting chickens, is caused by Avibacterium paragallinarum. The clinical manifestations of IC include nasal discharge, facial swelling, and lacrimation. This acute disease results in high morbidity and low mortality, while the course of the disease is prolonged and mortality rates are increased in cases with secondary infections. Studies regarding the immune response in infected chickens are scarce, and the local immune response is the focal point of investigation. However, a large body of work has demonstrated that severe infections can impact the systemic immune response. The objective of this study was to evaluate the systemic effects of Avibacterium paragallinarum (serovar B-1) infection on immune cells in specific pathogen-free (SPF) chickens. The current study revealed the presence of a transient circulating monocyte population endowed with high phagocytic ability and clear downregulation of major histocompatibility complex class II (MHC-II) surface expression. In human and mouse studies, this monocyte population (identified as tolerant monocytes) has been correlated with a dysfunctional immune response, increasing the risk of secondary infections and mortality. Consistent with this dysfunctional immune response, we demonstrate that B cells from infected chickens produced fewer antibodies than those from control chickens. Moreover, T cells isolated from the peripheral blood of infected chickens had a lower ability to proliferate in response to concanavalin A than those isolated from control chickens. These findings could be related to the severe clinical signs observed in complicated IC caused by the presence of secondary infections.

An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation.

BACKGROUND: In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry. RESULTS: Here, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction. CONCLUSIONS: In summary, we established a reliable protocol to evaluate the proliferation of CD4+ and CD8+ chicken T cells by flow cytometry. Moreover, as this is an in-house protocol, the cost per sample using this protocol is low, allowing its implementation in laboratories that process a large number of samples.

Draft Genome Sequence of Heavy Metal-Resistant Aeromonas veronii CTe-01, Isolated from a Peruvian Wastewater Treatment Plant.

Here, we report a draft genome sequence of Aeromonas veronii strain CTe-01 (4.5 Mb), a hemolytic, heavy metal-resistant bacterium isolated from a wastewater treatment plant located at Cachiche, Ica, Peru. These characteristics could be used for bioremediation of contaminated environments.

Genotype-matched Newcastle disease virus vaccine confers improved protection against genotype XII challenge: The importance of cytoplasmic tails in viral replication and vaccine design.

Although typical Newcastle disease virus (NDV) vaccines can prevent mortality, they are not effective in preventing viral shedding. To overcome this, genotype-matched vaccines have been proposed. To date, this approach has never been tested against genotype XII strains. In this study, we generated and assessed the protection against genotype XII challenge of two chimeric NDV vaccine strains (rLS1-XII-1 and rLS1-XII-2). The rLS1-XII-1 virus has the complete fusion protein (F) and the hemagglutinin-neuraminidase (HN) open reading frames replaced with those from genotype XII strain NDV/peacock/Peru/2011 (PP2011) in a recombinant LaSota (rLS1) backbone. In rLS1-XII-2 virus, cytoplasmic tails of F and HN proteins were restored to those of rLS1. In vitro evaluation showed that rLS1-XII-2 and the parental rLS1 strains replicate at higher efficiencies than rLS1-XII-1. In the first vaccine/challenge experiment, SPF chickens vaccinated with rLS1-XII-1 virus showed only 71.3% protection, whereas, rLS1 and rLS1-XII-2 vaccinated chickens were fully protected. In a second experiment, both rLS1-XII-2 and the commercial vaccine strain LaSota induced 100% protection. However, rLS1-XII-2 virus significantly reduced viral shedding, both in the number of shedding birds and in quantity of shed virus. In conclusion, we have developed a vaccine candidate capable of fully protecting chickens against genotype XII challenges. Furthermore, we have shown the importance of cytoplasmic tails in virus replication and vaccine competence.

Whole-Genome Sequencing of a Salmonella enterica subsp. enterica Serovar Infantis Strain Isolated from Broiler Chicken in Peru.

This report shows the whole-genome sequence of the multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis strain FARPER-219. Antibiotic resistance genes are found mainly in the plasmid. Our findings show important genetic information that provides an understanding of the recent spread of this serotype in poultry.

Evaluation of Plasmodium falciparum MSP10 and its development as a serological tool for the Peruvian Amazon region.

BACKGROUND: Different antigens are needed to characterize Plasmodium falciparum infection in terms of seroreactivity and targets for invasion inhibition, in order to guide and identify the proper use of such proteins as tools for the development of serological markers and/or as vaccine candidates. METHODS: IgG responses in 84 serum samples from individuals with P. falciparum infection [classified as symptomatic (Sym) or asymptomatic (Asym)], or acute Plasmodium vivax infection, from the Peruvian Amazon region, were evaluated by enzyme-linked immunosorbent assays specific for a baculovirus-produced recombinant protein P. falciparum Merozoite Surface Protein 10 (rMSP10) and for non-EGF region selected peptides of PfMSP10 selected by a bioinformatics tool (PfMSP10-1, PfMSP10-2 and PfMSP10-3). Monoclonal antibodies against the selected peptides were evaluated by western blotting, confocal microscopy and inhibition invasion assays. RESULTS: Seroreactivity analysis of the P. falciparum Sym- and Asym-infected individuals against rMSP10 showed a higher response as compared to the individuals with P. vivax acute infection. IgG responses against peptide PfMSP10-1 were weak. Interestingly high IgG response was found against peptide PfMSP10-2 and the combination of peptides PfMSP10-1 + PfMSP10-2. Monoclonal antibodies were capable of detecting native PfMSP10 on purified schizonts by western blot and confocal microscopy. A low percentage of inhibition of merozoite invasion of erythrocytes in vitro was observed when the monoclonal antibodies were compared with the control antibody against AMA-1 antigen. Further studies are needed to evaluate the role of PfMSP10 in the merozoite invasion. CONCLUSIONS: The rMSP10 and the PfMSP10-2 peptide synthesized for this study may be useful antigens for evaluation of P. falciparum malaria exposure in Sym and Asym individuals from the Peruvian Amazon region. Moreover, these antigens can be used for further investigation of the role of this protein in other malaria-endemic areas.

Glycoprotein G (gG) production profile during infectious laryngotracheitis virus (ILTV) infection.

Glycoprotein G (gG) is a conserved protein, and it has been described as a chemokine-binding protein in most members of the alphaherpesviruses. In case of the infectious laryngotracheitis virus (ILTV), an alphaherpesvirus that infects chickens, this protein is a virulence factor that plays an immunomodulatory role in the chicken immune response. Nevertheless, the gG production profile during ILTV infection has not yet been studied. In this study, we developed monoclonal antibodies in order to determine the gG production profile during ILTV infection in chicken hepatocellular carcinoma (LMH) cell cultures as well as embryonated specific-pathogen-free (SPF) chicken eggs and SPF chickens using a sandwich enzyme-linked immunosorbent assay (ELISA). Despite the fact that inoculated LMH cell cultures showed an increase in both gG production and viral genome copy number up to 96 h after inoculation, we observed that gG production started earlier than the increase in viral genome copy number in ILTV infected embryonated SPF chicken eggs. Likewise, a gG production peak and an increase of viral genome copy number was observed prior to the appearance of clinical signs in infected SPF chickens. According to the production profiles, gG was also produced quite early in eggs and chickens inoculated with ILTV. These findings contribute to the knowledge of the gG role during the ILTV infection as a virulence factor.

Genomic Islands in the Full-Genome Sequence of an NAD-Hemin-Independent Avibacterium paragallinarum Strain Isolated from Peru.

Here, we report the full-genome sequence of an NAD-hemin-independent Avibacterium paragallinarum serovar C-2 strain, FARPER-174, isolated from layer hens in Peru. This genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes.

Near-Complete Genome Sequence of Infectious Bronchitis Virus Strain VFAR-047 (GI-16 Lineage), Isolated in Peru.

Here, we report the near-complete genome sequence of the infectious bronchitis virus (IBV) strain VFAR-047, isolated in Peru in 2014. This strain was classified into GI lineage 16 (GI-16) based on both the genome and Spike 1 (S1) sequence analysis. Furthermore, four potential recombination events with other GI-16 and GI-11 strains were identified.

Whole-Genome Sequence of Sphingomonas sp. Strain FARSPH, a Novel Sf9 Insect Cell Culture Contaminant.

Here, we report the whole-genome sequence of Sphingomonas sp. strain FARSPH, isolated from an insect cell line as a contaminant. FARSPH shared high identity with Sphingomonas melonis and Sphingomonas aquatilis strains. Due to this finding, we recommend taking this genus into consideration for cell culture quality control.

Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

BACKGROUND: Infectious coryza (IC) is an acute respiratory disease of growing chickens and layers caused by Avibacterium paragallinarum. The development of tools that allow rapid pathogen detection is necessary in order to avoid disease dissemination and economic losses in poultry. An Av. paragallinarum-specific Ma-4 epitope of the TonB-dependent transporter (TBDT) was selected using bioinformatic tools in order to immunize a BalbC mouse and to produce monoclonal antibodies to be used in a lateral flow test (LFT) developed for Av. paragallinarum detection in chicken nasal mucus samples. RESULTS: The 1G7G8 monoclonal antibody was able to detect TBDT in Av. paragallinarum cultures (serogroups: A, B and C) by Western blot and indirect ELISA assay. Consequently, we developed a self-pairing prototype LFT. The limit of detection of the prototype LFT using Av. paragallinarum cultures was 1 × 104 colony-forming units (CFU)/mL. Thirty-five nasal mucus samples from chickens suspected of having infectious coryza were evaluated for the LFT detection capacity and compared with bacterial isolation (B.I) and polymerase chain reaction (PCR). Comparative indicators such as sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive values (NPV) and the kappa index (K) were obtained. The values were 100.0% Se, 50% Sp, 65.4% PPV, 100% NPV, and 0.49 K and 83.9% Se, 100% Sp, 100% PPV, 44.4% NPV, and 0.54 K for the comparison of the LFT with B.I and PCR, respectively. Additionally, the LFT allowed the detection of Av. paragallinarum from coinfection cases of Av. paragallinarum with Gallibacterium anatis. CONCLUSIONS: The results indicate that the self-pairing prototype LFT is suitable for the detection of TBDT in Av. paragallinarum cultures as well as in field samples such as nasal mucus from Av. paragallinarum-infected chickens. Therefore, this prototype LFT could be considered a rapid and promising tool to be used in farm conditions for Av. paragallinarum diagnosis.

Full-Genome Sequence of Infectious Laryngotracheitis Virus (Gallid Alphaherpesvirus 1) Strain VFAR-043, Isolated in Peru.

We report here the first genome sequence of infectious laryngotracheitis virus isolated in Peru from tracheal tissues of layer chickens. The genome showed 99.98% identity to the J2 strain genome sequence. Single nucleotide polymorphisms were detected in five gene-coding sequences related to vaccine development, virus attachment, and viral immune evasion.