RESUMO
Avian influenza virus (AIV) represents a major concern with productive implications in poultry systems but it is also a zoonotic agent that possesses an intrinsic pandemic risk. AIV is an enveloped, negative-sense and single-stranded RNA virus with a segmented genome. The eight genomic segments, comprising the whole genome, encode for eleven proteins. Within these proteins, Hemagglutinin (HA) and Neuraminidase (NA) are the most relevant for studies of evolution and pathogenesis considering their role in viral replication, and have also been used for classification purposes. Migratory birds are the main hosts and play a pivotal role in viral evolution and dissemination due to their migratory routes that comprise large regions worldwide. Altogether, viral and reservoir factors contribute to the emergence of avian influenza viruses with novel features and pathogenic potentials. The study aimed to conduct surveillance of AIVs in wild birds from Peru. A multi-site screening of feces of migratory birds was performed to isolate viruses and to characterize the whole genome sequences, especially the genes coding for HA and NA proteins. Four-hundred-twenty-one (421) fecal samples, collected between March 2019 and March 2020 in Lima, were obtained from 21 species of wild birds. From these, we isolated five AIV from whimbrel, kelp gull, Franklin's gulls and Mallard, which were of low pathogenicity, including four subtypes as H6N8, H13N6, H6N2 and H2N6. Genetic analysis of HA and NA genes revealed novel features in these viruses and phylogenetic analysis exhibited a close relationship with those identified in North America (US and Canada). Furthermore, H2N6 isolate presented a NA sequence with higher genetic relationship to Chilean isolates. These results highlight that the geographical factor is of major relevance in the evolution of AIV, suggesting that AIV circulating in Peru might represent a new site for the emergence of reassortant AIVs.
Assuntos
Charadriiformes , Vírus da Influenza A , Influenza Aviária , Animais , Animais Selvagens , Aves , Hemaglutininas/genética , Neuraminidase/genética , Peru/epidemiologia , FilogeniaRESUMO
The coronavirus disease-19 (COVID-19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine-resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice and hamsters. Intranasal administration of rNDV-based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV-based vaccine candidates fully protected animals against SARS-CoV-2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID-19.
Assuntos
COVID-19 , Vacinas Virais , Administração Intranasal , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Cricetinae , Camundongos , Vírus da Doença de Newcastle/genética , SARS-CoV-2/genética , Vacinação , Vacinas Sintéticas/genéticaRESUMO
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.
Assuntos
Doença de Newcastle/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Genótipo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Virulência/genética , Virulência/imunologia , Replicação Viral/genética , Replicação Viral/imunologia , Eliminação de Partículas Virais/genética , Eliminação de Partículas Virais/imunologiaRESUMO
BACKGROUND: Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV. METHODS: In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP. RESULTS: The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R 2 = 0.816) and ELISA (R 2 = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R 2 = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs. CONCLUSIONS: Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.
Assuntos
Galinhas , Testes de Neutralização/métodos , Testes de Neutralização/normas , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Testes de Inibição da Hemaglutinação , Doença de Newcastle/sangue , Doença de Newcastle/imunologia , Reprodutibilidade dos Testes , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologiaRESUMO
BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.