Construction of recombinant adenovirus expression vector of avian reovirus σC protein and its effect on proliferation of hepatocellular carcinoma cells / 中国生物制品学杂志

@#Objective To construct the recombinant adenovirus vector pAd-σC for the expression of avian reovirus(ARV)aC protein and to detect its effects on the proliferation of hepatocellular carcinoma cells,in order to build up a basis for the development of novel anti-tumor vaccines.Methods The recombinant shuttle vector pShuttle-σC was constructed by PCR amplification of ARV σC gene,and then transformed into competent BJ5183 cells containing the adenovirus vector pAdessy-1.The recombi-nant adenovirus vector pAd-σC was obtained by homologous recombination,and the virus was packaged in HEK293 cells.The virus titer was measured by TCID_(50),the expression of σC protein was determined by Western blot and ELISA,and the effect of virus on the proliferation of human hepatocellular carcinoma cells SMMC7721 was detected by CCK-8 assay.Results The recombinant shuttle vector pShuttle-σC was confirmed to be constructed correctly by double enzyme digestion and sequen-cing,and the recombinant adenovirus vector pAd-σC was constructed correctly as identified by colony PCR.σC protein was successfully expressed in hepatocellular carcinoma cells SMMC7721.The recombinant adenovirus Ad-σC had a titer of 10~(7.5)/0.1 mL,which inhibited the proliferation of hepatocellular carcinoma cells SMMC7721.Conclusion The recombinant adenovirus vector pAd-trC containing ARV σC gene was successfully constructed,and its inhibitory effect on tumor cell proliferation was preliminarily analyzed,which lays a foundation for revealing the molecular mechanism of ARV oncolytic effect and further developing novel anti-tumor biological preparation.

Influenza aviar H5N1. ¿Debemos preocuparnos?

Desde la segunda mitad de 2022 se ha reportado un aumento de casos de influenza en aves migratorias en Latinoamérica. Los virus influenza A y B son los principales agentes asociados a influenza estacional epidémica en humanos. Los virus influenza A circulan no solo en humanos sino también en animales, incluyendo aves migratorias. El intercambio de segmentos de ARN genómico entre dos virus del mismo tipo aumenta la diversidad de los subtipos circulantes e incluso puede facilitar la generación de progenie viral potencialmente pandémica. La naturaleza zoonótica del virus influenza A puede generar infecciones en humanos con virus de origen animal. El virus influenza A de origen aviar ha ocasionado transmisiones en humanos, incluyendo casos graves y muertes, siendo la influenza A H5N1 la más destacada. Es importante tomar medidas de prevención y control en caso de aumento de casos de influenza en aves migratorias para prevenir posibles pandemias en Chile y el mundo.

Since the second half of 2022, an increase in influenza cases in migratory birds has been reported in Latin America. Influenza A and B viruses are the main agents associated with seasonal epidemic influenza in humans. Influenza A viruses circulate not only in humans but also in animals, including migratory birds. The exchange of genomic RNA segments among two viruses increases the diversity of circulating subtypes and may even facilitate the generation of potentially pandemic viral progeny. The zoonotic nature of influenza A virus can generate infections in humans with animal-origin viruses. Avian-origin influenza A virus has caused transmissions in humans, including severe cases and deaths, with influenza A H5N1 being the most prominent. It is important to take preventive and control measures in case of an increase in influenza cases in migratory birds to prevent possible pandemics in Chile and the world.

Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering / 生物工程学报

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Molecular characterization and phylogenetic analysis of avian influenza H3N8 virus isolated from imported waterfowl in Malaysia

@#Wild aquatic birds are natural reservoirs of influenza A viruses and H3 subtype is one of the most prevalent subtypes in waterfowl. Two H3N8 viruses of low pathogenic avian influenza (LPAI) were isolated via egg inoculation technique from the fecal swab specimens from imported barnacle goose and paradise shelduck in Veterinary Research Institute Ipoh, Malaysia. The full length of eight gene segments of the two viruses were amplified and sequenced with specific primers. The sequences were molecularly characterized, and the sequence identity were assessed with other published sequences. The two viruses are identical and they possess the same amino acid sequences for all the eight gene segments. The viruses were highly similar to the H3 virus from Netherlands and N8 virus from Belgium respectively. Phylogenetic analysis revealed that all the eight gene segments were grouped in the Eurasian lineage, and genetic reassortment may occur between the internal genes of the H3 viruses and other AI subtypes. Though four amino acid substitutions were identified in the hemagglutinin gene, the viruses retained most of the avian-type receptor binding preference. Few amino acid substitutions were observed in all internal genes. Most of the neuraminidase inhibitors and adamantine resistance related mutation were not seen in the viruses. The replicative capacity, cross species transmissibility, and potential zoonotic risk of the viruses are worth further investigation. As H3 virus poses potential threats to both human and animals, and with the increase in the international trade of birds; strict quarantine practice at the entry point and good laboratory diagnostic capabilities is crucial to prevent the introduction of new AI virus into our country.

Surveillance and analysis of avian influenza viruses in the external environment of poultry in Anqing City， Anhui Province / 上海预防医学

ObjectiveTo determine the prevalence of avian influenza viruses in the external environment of poultry in Anqing City， Anhui Province， and provide scientific evidence for prevention and control of animal-derived influenza in humans. MethodsA total of 28 farmers’ markets/farms in 10 counties （cities， districts） of Anqing City， Anhui Province， were selected as surveillance sites by simple random sampling strategy. Poultry faeces and other related samples were collected for 6 consecutive weeks. Real-time fluorescence quantitative PCR was used to examine the nucleic acids of influenza A virus. Subtypes H5， H7， and H9 of avian influenza virus were further tested in the positive samples. ResultsA total of 426 specimens were collected， among which 113 tested positive with a positive rate of 26.53%. Among the positive specimens， 104 were determined to be subtype H9， accounting for 92.04%. It did not significantly differ in the positive rate between the main and non-main urban areas （χ2<0.01， P>0.05） or among the specimens collected in different weeks （χ2=7.57， P>0.05）. However， it significantly differed in the positive rate among the specimens collected in the third week and other weeks （χ2=6.89， P<0.05）. Furthermore， among the different sampling sites， farms had the highest positive rate of 46.67%. Among the specimens from different sources， the surface-coated specimens from poultry cages had the highest positive rate of 34.78%. ConclusionAvian influenza viruses are prevalent in the external environment of poultry in Anqing City. It warrants strengthening the surveillance and risk assessment to reduce the virus transmission in the external environment and risk of human infection with animal-derived influenza.

Sequence analysis of HA and NA genes of human infected H9N2 avian influenza virus in Yunnan province, 2019 / 中国热带医学

@#Abstract: Objective To investigate the molecular characteristics of the H9N2 avian influenza virus (AIV) causing human infection in Yunnan Province in 2019, and to provide the scientific basis for the prevention and control of avian influenza in Yunnan Province. Methods Influenza virus typing was performed by real-time RT-PCR in two influenza-like illness samples, and the Illumina Miseq high-throughput sequencer was used to determine the viral genome sequence. HA and NA gene sequence alignment and phylogenetic tree construction were performed using Mega7.0 software. Results Real-time RT-PCR results showed that two influenza-like illness samples were positive for H9N2 subtype. The full length of HA and NA were obtained by genomic sequencing. Sequence system evolution analysis showed that the HA and NA of the two AIVs in Yunnan Province were in the same evolutionary clade as A/Chicken/Zhejiang/HJ/2007 and belonged to the G57 type. The HA nucleotide and amino acid homology of the two AIVs were 93.92% and 95.00%, respectively, and the NA nucleotide and amino acid homology was 93.31% and 82.03%, respectively. The nucleotide (amino acid) homology of HA was 92.29%-96.94% (93.77%-98.43%) and 92.84%-94.92% (94.18%-96.23%), respectively, and NA nucleotide homology (amino acid) were 91.81%-97.60% (77.82%-94.83%), 94.38%-97.22% (85.47%-94.55%), respectively, compared with that of human infected H9N2 epidemic strains obtained in China from 2015 to 2020. Both AIVs HA protein cleavage site sequences were PSRSSR↓GLF, which was in line with the characteristics of low pathogenic influenza. The analysis of HA protein receptor binding site showed that amino acids at positions 109, 161, 163, 191, 202, 203 and 234 were consistent with the reference strains, while amino acids at position 198 were mutated to T. N166D and 168N mutations were also found in HA protein, and both AIVs had 7 potential glycosylation sites. Analysis of the erythrocyte binding site of NA gene found that there were amino acid mutations at positions 369, 402, 403, and 432, and amino acid deletion at positions 63-65 was found in the NA genes. There were 4 and 5 potential glycosylation sites in the two AIVs, respectively, and no drug resistance site mutations were found. Conclusions　The receptor binding sites, erythrocyte binding sites and glycosylation sites of HA and NA genes of H9N2 AIV in Yunnan Province have different degrees of variation, and monitoring and prevention and control should be strengthened.

Efficacy of some avian influenza H5 vaccines against local highly pathogenic avian influenza viruses subtype H5N8 isolated in 2018 and 2020 in Egypt

Commercial inactivated avian influenza H5 vaccine is used as an essential control strategy for avian influenza disease in Egypt. Since the initial outbreaks of highly pathogenic avian influenza H5N8, the virus has diverged with new genotypes and variant viruses continuing to emerge which mainly stand behind vaccination failure. In the present work, four different commercial avian influenza vaccines were inoculated in specific pathogenic free chickens for assessing its efficacy against local highly pathogenic avian influenza H5N8 virus isolated in 2018 and 2020. Two hundred and forty specific pathogenic free chickens were clustered into four groups; each group was inoculated with the corresponding vaccine (60 specific pathogenic free chickens/vaccine). Sixty specific pathogenic free chicks were kept as control unvaccinated group. Sera collected from vaccinated chicken groups at 3rd and 4th week post vaccination were examined for calculating neutralizing antibodies using heterologous highly pathogenic avian influenza H5N8 2018 and 2020. At 4th week post vaccination, vaccinated chickens were challenged; moreover, oropharyngeal swabs were collected from challenged vaccinated chickens to calculate the viral shedding. Our findings revealed the groups vaccinated with vaccine code no 1 and 2 that contains two vaccine strains (H5N1 and H5N8) of local origin exhibited the highest hemagglutination inhibition titer, protection (percent) and reduction in viral shedding titer when examined by highly pathogenic avian influenza H5N8 2018 while, vaccine code no 3 induced lower antibody response, protection (percent) and reduction in viral shedding, but still within satisfactory level when compared to previous groups. When highly pathogenic avian influenza H5N8 2020 was used, it was found the seroconversion rate, protection (percent) and mean titer of reduction of viral shedding decreased in comparison to those recorded for highly pathogenic avian influenza H5N8 2018. Vaccine code no 4 was impotent to either highly pathogenic avian influenza 2018 or 2020. Accordingly, it was recommended to update vaccine strain according to epidemiological condition and used the predominant circulating strain isolate in challenge test(AU)

La vacuna comercial inactivada H5 se utiliza como estrategia esencial de control de la enfermedad de la gripe aviar en Egipto. Desde los brotes iniciales de la gripe aviar altamente patógena H5N8, el virus ha variado al aparecer continuamente nuevos genotipos y variantes virales, que son los principales responsables del fracaso de la vacunación. En el presente trabajo, cuatro vacunas comerciales diferentes contra la gripe aviar se inocularon en pollos libres de patógenos específicos para evaluar su eficacia contra cepas del virus local de la gripe aviar altamente patógeno H5N8 aisladas en 2018 y 2020. Se agruparon 240 pollos pollos libres de patógenos específicos en cuatro grupos, cada uno fue inoculado con la vacuna correspondiente (60 pollos pollos libres de patógenos específicos/vacuna). Sesenta pollos SPF se mantuvieron como grupo control sin vacunar. Los sueros de los pollos vacunados recogidos en la 3ª y 4ª semana después de la vacunación se examinaron para calcular los anticuerpos neutralizantes contra la gripe aviar heteróloga H5N8 2018 y 2020. En la cuarta semana después de la vacunación, los pollos vacunados fueron retados; además, se recogieron hisopados orofaríngeos de los pollos vacunados retados para calcular la diseminación viral. Nuestros resultados revelaron que los grupos vacunados con las vacunas con códigos nº 1 y 2, que contienen dos cepas vacunales (H5N1 y H5N8) de origen local, mostraron el mayor título de inhibición de la hemaglutinación, protección (por ciento) y reducción del título de excreción viral cuando se evaluaron contra la gripe aviar altamente patógena H5N8 2018, mientras que la vacuna con código nº 3 indujo menor respuesta de anticuerpos, protección (por ciento) y reducción de la excreción viral, pero todavía dentro de un nivel satisfactorio en comparación con los grupos anteriores. Al utilizar la vacuna contra la gripe aviar altamente patógena H5N8 2020, se observó que la tasa de seronconversión, la protección (por ciento) y el título medio de reducción de la excreción viral disminuyeron en comparación con los registrados para la gripe aviar altamente patógena H5N8 2018. La vacuna con código nº 4 no fue potente para la gripe aviar altamente patógena de 2018 o de 2020. Por consiguiente, se recomendó actualizar la cepa de la vacuna de acuerdo con las condiciones epidemiológicas y utilizar el aislamiento de la cepa circulante predominante en la prueba de reto(AU)

Impact of H5N8 duck and chicken viral isolates on the immunological profile of vaccinated specific pathogen free chickens

Nowadays, there is a global concern about outbreaks caused by the highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 which caused devastating losses in the poultry industry sector. This clade was subdivided into two waves: clade 2.3.4.4A from 2014 to 2015 and clade 2.3.4.4b from 2016 until now. In this literature we aimed to evaluate the efficacy of recently used inactivated commercial avian influenza vaccines against two new Egyptian highly pathogenic avian influenza virus H5N8 isolates of clade 2.3.4.4b, A/chicken/Egypt/1526v/2020/H5N8 (H5N8-CH) and A/Duck/Egypt/Qalubia321/2021 (H5N8-D). Three-week-old specific pathogen free chickens were vaccinated with eight types of the most recently used inactivated avian influenza vaccines containing homologous and heterologous virus to the circulating H5N8 isolates. All specific pathogen free chicken groups were bled weekly post vaccination for antibody analysis using two H5N8 isolates of chicken and duck origin as antigen in hemagglutination inhibition test. Also, all vaccinated chicken groups were challenged 4 weeks post vaccination against the H5N8 duck isolate with a dose of 109 EID50/0.1 mL per chicken to measure the protection percentage of the commercial vaccines used. The results showed that vaccines with homologous and heterologous virus showed variable degrees of accepted protection percentage ranged from 90percent to 100percent, thus it was concluded that not only the genetic and antigenic match of the vaccine strains with the circulating highly pathogenic avian influenza viruses influences vaccine efficiency; other factors, such as manufacturing procedures, adjuvant, antigen content, vaccine dose and administration factors could affect vaccine efficacy, therefore, further vaccine development studies are needed to improve the percentage of protection and prevention of viral shedding against local highly pathogenic avian influenza H5 viruses in Egypt(AU)

En la actualidad, existe una preocupación mundial por los brotes causados por el virus de la gripe aviar altamente patógena H5N8 clado 2.3.4.4 que causó pérdidas devastadoras en el sector de la industria avícola. Este clado se subdividió en dos oleadas: clado 2.3.4.4A de 2014 a 2015 y clado 2.3.4.4b de 2016 hasta ahora. En el presente trabajo, dos aislamientos egipcios de la gripe aviar altamente patógena H5N8 del clado 2.3.4.4b, A/chicken/Egypt/1526v/2020/H5N8 (H5N8_CH) y A/Duck/Egypt/Qalubia321/2021 (H5N8_D), se utilizaron para evaluar la eficacia de vacunas comerciales inactivadas contra la gripe aviar de reciente utilización. Pollos libres de patógenos específicos de tres semanas de edad fueron vacunados con ocho vacunas inactivadas contra la influenza aviar, de uso reciente, que contenían virus homólogos y heterólogos a los aislamientos circulantes de H5N8. Todos los grupos de pollos libres de patógenos específicos fueron sangrados semanalmente tras la vacunación para el análisis de anticuerpos; dos virus H5N8 aislados de pollo y pato se utilizaron como antígeno en la prueba de inhibición de la hemaglutinación. Además, todos los grupos de pollos vacunados fueron retados 4 semanas después de la vacunación con el virus H5N8 aislado de pato, con una dosis de 109 EID50/0,1 mL por pollo, para medir el porcentaje de protección de las vacunas comerciales utilizadas. Los resultados mostraron que las vacunas con virus homólogos y heterólogos presentaron grados variables de aceptada protección, la que osciló entre el 90 por ciento y el 100 por ciento, por lo que se concluyó que no sólo la coincidencia genética y antigénica de las cepas vacunales con los virus circulantes de la influenza aviar altamente patógena influye en la eficacia de la vacuna; otros factores, como los procedimientos de fabricación, el adyuvante, el contenido en antígenos, la dosis de la vacuna y los factores de administración podrían afectar a la eficacia de la vacuna, por lo que es necesario seguir estudiando el desarrollo de vacunas para mejorar la protección y la prevención de la excreción viral contra los virus H5 de la influenza aviar altamente patógena locales en Egipto(AU)

Bird Migration with Visual Avian Navigation & Nest Nidification: The Spatial Linear Geometries Georeferencing Functionality

Problem: Bird migration (eye): Georeferencing procedure with clues, rules, functionalities, and restrictions, for avian navigation and nest nidification.Literature Knowledge: Computer vision (sensor): Robot self-referencing with the Perspective-n- Point pose estimation technique.Aim: Hypothesis introduction and proving (“The birds also follow the same georeferencing procedure like robots in avian navigation and nest nidification”).Methodology: (a) Reference data, images, and photography acquisition and 4-means layering (eBird dataset, Flickr imagery, CORINE land covering, and Volunteered Geographic Information);(b) Image processing; and (c) GIS spatial overlay analysis.Results: Statistical spatial analysis using data of the GIS overlays (the 4 layers). Correlation matrix (Avian navigation and nest nidification in low-density urban areas as these are affected by spatial linear geometries and land cover types).Conclusion: A statistically satisfactory approach to the introduced hypothesis.Potential Applications: Human spatial cognition and movement behavior; Children’s motor control and coordination.

Selection of Lactic Acid Bacteria Isolated from Cocoa and Cassava Fermentation as Potential Probiotic for Pathogenic Microorganisms Control in Poultry Farming

Aims: The overuse of antibiotics in animal farming sector is leading to an increase drug resistant bacteria rate. This situation makes it difficult to treat pathologies in both humans and animals. The aim of this study was to assess some probiotic profiles of lactic acid bacteria strains isolated from cocoa fermentation and traditional cassava ferment for possible use as potentially probiotic strains for the monitoring of pathogenic microorganisms in poultry farming. Methodology: Thus, a total of 267 lactic acid bacteria strains were tested for analysis of the antibacterial activity against the growth capacity of Salmonella and E. coli isolates. Probiotic properties of Lactic acid bacteria were consisted of acidification capacity, resistance to acid shock and to salt bile containing in culture medium and capacity to produce proteolytic and lipolytic enzymes. Results: Among them, 25 strains have induced the high bacterial growth inhibition against these pathogenic bacteria with inhibition zone diameters ranged from 9 to 27 mm. Among these strains, 20 isolates showed high resistance to acid shock at pH ? 4 and six strains were able to grow at pH 3.5 with survival rate range from 30 % to 89 %. Moreover, six of these strains, including four isolates of Lactobacillus plantarum (T1GB8, T11AB17, LAB26, LAB 127), one strain of Leuconostoc mesenteroides (T0AB9) and one isolate of Enterococcus facium (LAB18), have shown capacity to growth with 1 % of bile salts in the medium. Even better, these strains exhibited capacity to produce proteolytic and lipolytic enzymes with halo around the well diameters reached 29 mm for some strains. Conclusion: This study shows the possibility of use probiotics lactic acids bacteria as antibiotics alternative in poultry sector to reduce some avian pathologies affecting the poultry sector in C魌e d'Ivoire.

Identification and phylogenetic analysis of five highly pathogenic avian influenza (H5N8) viruses isolated in Urumqi in 2016 / 中华微生物学和免疫学杂志

Objective:To analyze the genetic evolution and molecular characteristics of H5N8 avian influenza viruses (AIVs) isolated from the poultry in a live poultry market (LPM) in Urumqi, Xinjiang.Methods:Oropharyngeal and cloacal swabs of poultry were collected from a LPM in Urumqi in 2016. AIVs were isolated by inoculating swab samples into chicken embryos. Hemagglutination test and RT-PCR were used to identify the AIVs. The genes of isolated AIVs were amplified with the universal primers of AIV and whole-genome sequencing was also performed. Pairwise sequence alignment and analysis of phylogenetic and molecular characteristics were performed using BLAST, Clustal W, MEGA-X and DNAStar software.Results:Five H5N8 AIVs were isolated from poultry. These strains shared a nucleotide identity of 99.70%-100.00%, which indicated that they were from the same source, and were named XJ-H5N8/2016. Phylogenetic analysis based on hemagglutinin( HA), NS and PB2 genes showed that these isolates were clustered together with H5N8 AIVs isolated from the migratory swans in Hubei, Shanxi and Sanmenxia, and the ducks in India during 2016 to 2017. Moreover, they were also clustered together with H5N6 AIVs isolated from minks in China and the first case of human infection in Fujian. The phylogenetic tree of neuraminidase( NA) gene indicated the five isolates clustered together with H5N8 AIVs isolated from ducks in India in 2016, and the phylogenetic trees of PB1, MP, PA and NP genes showed that they were clustered together with H5N8 AIVs isolated from wild birds and poultry in Egypt, Cameroon, Uganda, Congo and other African countries in 2017. The HA cleavage sites of XJ-H5N8/2016 contained five consecutive basic amino acids, indicating high pathogenicity. Multiple mutations in the genes of XJ-H5N8/2016 could enhance its virulence and pathogenicity to mammals. Conclusions:The five strains of H5N8 AIVs isolated from the LPM were highly pathogenic and closely related to the H5N8 AIVs isolated from migratory birds and poultry in Hubei, Shanxi, Sanmenxia area, Africa and India during 2016 to 2017. Meanwhile, some of the viral genes were also closely related to the H5N6 AIVs isolated from the minks and human in China. Multiple mutations could increase the virulence and pathogenicity of AIVs to mammals, which could pose a potential threat to public health.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus / 生物工程学报

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

Ruptura del paradigma: estudio macroscópico del desarrollo embrionario de aves (Gallus gallus domesticus) realizado por métodos complementarios / Paradigm break: macroscopic study of embryonic development of birds (Gallus gallus domesticus) carried out by complementary methods

RESUMEN: El uso de plataformas virtuales se muestra como un nuevo recurso didáctico que posibilita el proceso de enseñanza-aprendizaje de forma dinámica. A grandes rasgos, permite el acceso a imágenes digitales en alta resolución mediante el uso de computadores, smartphones y/o tabletas. Portanto, este trabajo presenta nuestra metodología en el campo de la embriología de aves domésticas y la experiencia adquirida en el desarrollo de recursos para la enseñanza por medio de las tecnologías de la información y comunicación, de gran utilidad hoy en día en medio de la pandemia ocasionada por el nuevo coronavirus.

SUMMARY: Online platforms are a new didactic resource that enable an active teaching-learning process. In general, they allows access to high resolution digital images through the use of computers, smartphones and / or tablets. Therefore, this study presents our methodology in the field of domestic bird embryology and the experience acquired in the development of teaching resources through information and communication technologies, which are very useful nowadays, particularly in the midst of the pandemic caused by the new coronavirus.

Haemoparasites in captive birds at Uberlândia zoo, state of Minas Gerais, Brazil

Haemoparasites are one of the most important groups of bird parasites, with emphasis on the genera Haemoproteus, Plasmodium, Leucocytozoon and Trypanosoma. Zoos sustain different wild animals and are valuable tools for the education and conservation of species. The conditions of captive animals differ from wild animals, as zoos have sufficient availability of food throughout the year, protection against predators and veterinary care for animals. This study aimed to determine the prevalence of haemoparasites in captive birds of the Sabiá Municipal Park Zoo, municipality of Uberlândia, state of Minas Gerais, Brazil. Blood samples were collected from the alveolar vein puncture to make blood swabs. This material was fixed with methanol, stained by the GIEMSA technique and examined under optical microscope. A total of 46 birds (19 species) were analyzed and only three individuals (6.52%) were positive for Plasmodium sp. The hosts were Pavo cristatus and Tyto furcata. This low positivity was expected, since haemoparasites do not generally present high infection rates among birds. Even if a parasite is not pathogenic for a given species, these individuals are important reservoirs for the infection of more vulnerable species. Differences in the prevalence and intensity of infection of these hosts depend on the virulence of the parasite, ability of the host to respond to such infections and vector availability. This low prevalence rate suggests a good health status of the captive birds in the study area.

Diferencias entre el SARS-CoV-2 y el coronavirus de la Bronquitis Infecciosa Aviar (IBV) / Differences between SARS-CoV-2 and Avian Infectious Bronchitis (IBV) coronavirus / Diferenças entre o coronavírus SARS-CoV-2 e Bronquite Infecciosa Aviária (IBV)

Resumen La enfermedad por el nuevo coronavirus 2019 (COVID-19) es causada por el virus denominado SARS-CoV-2, no obstante, en los pollos de corral los coronavirus causan Bronquitis Infecciosa Aviar. En la actualidad, se ha logrado analizar la secuencia genómica del virus SARS-CoV-2, el cual indica que éste emergió de un reservorio animal, incluso se ha considerado que un virus aislado de un murciélago, idéntico a SARS-CoV, sea el progenitor del nuevo coronavirus. En otros estudios, se ha evidenciado que la glicoproteína de la espícula viral tiene un alto grado de emparentamiento entre virus que infectan mamíferos y aves, que es la que permite el contacto con el huésped. Mientras que en el caso del IBV, al inhalarse, el virus se ligará a los receptores de glucoproteína que contienen ácido siálico en las células epiteliales ciliadas del tejido respiratorio, entonces la replicación viral dará como resultado la pérdida de la función ciliar, acopiamiento de moco, necrosis y descamación, provocando dificultad para respirar y asfixia. El IBV afecta tráquea, riñones y tracto reproductivo de muchas aves. En el caso de las gallinas, el IBV virémico causa lesiones en el magnum y en el útero. Esta revisión dilucida algunos puntos clave en las diferencias en el nuevo coronavirus y el virus de la bronquitis infecciosa. Es muy poco probable que SARS-CoV-2 infecte o cause enfermedades en las aves de corral.

Abstract The new coronavirus 2019 disease (COVID-19) is caused by the virus called SARS-CoV-2, however, in free-range chicken's coronaviruses cause Avian Infectious Bronchitis. Currently, it has been possible to analyze the genomic sequence of the SARS-CoV-2 virus, which indicates that it emerged from an animal reservoir, it has even been considered that a virus isolated from a bat, identical to SARS-CoV, is the parent of the new coronavirus. In other studies, it has been shown that the glycoprotein of the viral spicule has a high degree of relationship between viruses that infect mammals and birds, which is the one that allows contact with the host. Whereas in the case of IBV, when inhaled, the virus will bind to sialic acid-containing glycoprotein receptors in hair epithelial cells of respiratory tissue, then viral replication will result in loss of ciliary function, mucus clearance, necrosis, and peeling, causing shortness of breath and suffocation. IBV affects the trachea, kidneys, and reproductive tract of many birds. In chickens, viremic IBV causes lesions in the magnum and the uterus. This review elucidates some key points in the differences between the novel coronavirus and the infectious bronchitis virus. SARS-CoV-2 is highly unlikely to infect or cause disease in poultry.

Resumo A nova doença coronavírus 2019 (COVID-19) é causada pelo vírus denominado SARS-CoV-2, no entanto, em galinhas caipiras, os coronavírus causam bronquite infecciosa aviária. Atualmente, foi possível analisar a sequência genômica do vírus SARS-CoV-2, o que indica que ele emergiu de um reservatório animal, inclusive se considerou que um vírus isolado de um morcego, idêntico ao SARS-CoV, é o progenitor do novo coronavírus. Em outros estudos, foi demonstrado que a glicoproteína da espícula viral possui alto grau de parentesco entre os vírus que infectam mamíferos e aves, o que permite o contato com o hospedeiro. Enquanto no caso do IBV, quando inalado, o vírus se liga aos receptores de glicoproteína contendo ácido siálico nas células epiteliais ciliadas do tecido respiratório, a replicação viral resultará em perda da função ciliar, acúmulo de muco , necrose e descamação, causando falta de ar e sufocação. O IBV afeta a traqueia, os rins e o trato reprodutivo de muitas aves. No caso das galinhas, o IBV virêmico causa lesões no magno e no útero. Esta revisão elucida alguns pontos-chave nas diferenças entre o novo coronavírus e o vírus da bronquite infecciosa. É altamente improvável que o SARS-CoV-2 infecte ou cause doenças em aves.

Effect of compound houttuynia mixture on influenza virus in vitro / 药学学报

Compound houttuynia mixture belongs to OTC class A medicine, which is made from Houttuynia cordata, Scutellaria baicalensis, Radix Isatidis, Forsythia, and Lonicera. As a kind of compound preparation of traditional Chinese medicine, houttuynia cordata mixture has extensive pharmacological effects, for example, clearing away heat and detoxifying, thus it is used for the sore throat, acute pharyngitis, and tonsillitis with wind-heat syndrome. In this study, the antiviral activity against influenza viruses and the primary mechanism of compound houttuynia mixture was evaluated. The antiviral effect of compound houttuynia mixture was determined by cytopathic effects (CPE), Western blot, quantitive reverse transcription PCR (qRT-PCR), and virus titer assays. The effect of houttuynia mixture on the replication cycle of influenza virus was evaluated by time-of-addition assay. In conclusion, the results showed that the compound houttuynia mixture had a broad-spectrum effect against influenza virus, including the international common influenza virus strains, the drug-resistant strains and the highly pathogenic avian influenza viruses H5N1 and H7N9. It mainly impairs the early stage of the viral replication.

Pathological, microbiological and immunohistochemical characterization of avian colibacillosis in broiler chickens of Mozambique / Caracterização patológica, microbiológica e imuno-histoquímica de colibacilose em frangos de corte de Moçambique

Avian colibacillosis is an acute and globally occurring infectious disease of domestic and wild birds caused by Escherichia coli, and it is associated with considerable economic losses mainly due to the morbidity and mortality associated. The present study aimed to describe the pathological, bacteriological and immunohistochemical aspects of avian colibacillosis in broiler chickens of Mozambique. Forty-nine broiler chicken presented anorexia, decreased weight gain, ataxia, diarrhea, dyspnea, and death in a clinical course of 3-5 days. The birds were raised in five farms (small, medium and large farms) with manual and automatic breeding system, with flocks ranging from 100 to 20,000 birds. At the necropsy, all birds had poor body condition, and the pericardium and the Glisson's capsule of all avian exhibited different degrees of adherence often associated with severe fibrin deposition. The thoracic and abdominal air sacs were thickened and adhered to the costal wall. Mild, moderate or marked hepatomegaly associated with white pinpoint multifocal areas (100%, 49/49) and mild to moderate splenomegaly in 75.5% (37/49) with a mottled surface were observed. The lungs and kidney were enlarged and reddish. Histologically, a multiorgan fibrinoheterophilic polyserositis was observed in 75.5% of the cases (37/49), which were characterized by inflammatory infiltrates composed mainly of degenerative heterophils, macrophages and plasma cells, associated with fibrin deposits and intermixed by coccobacillary bacterial basophilic aggregates. These affected mainly the pericardium (28.6%, 14/49), the pleura (18.4%, 9/49), the Glisson's capsule (10.2%, 5/49), the ventriculus (10.2, 5/33), and the proventriculus (8.2%, 4/49) serosa. Multifocal to coalescing areas of coagulative necrosis associated with similar inflammatory cells were observed mainly in the spleen (28.6%, 14/49), liver (24.5%, 12/49), and intestines (22.4%, 11/49). A similar infiltrate was also observed affecting the the lungs (16.3%, 8/49), the kidney (16.3%, 8/49) and the myocardium (14.3%, 7/49). Isolation and identification of E. coli was obtained in 12 cases through bacterial culture. Some organs (2 cases of each farms) were selected and submitted to immunohistochemistry anti-E. coli, and a positive stain was observed in all tested cases in liver (3/3), heart (4/4), spleen (1/1), lungs (4/4), intestines (4/4), bursa of Fabricius (1/1), ventriculus (1/1), and proventriculus (1/1) tissue sections. These results demonstrate that E. coli was the cause of mortality in these birds. Therefore, biosecurity and management measures should be employed to prevent and control the disease occurrence in Mozambique's poultry farming.(AU)

A colibacilose aviária é uma doença aguda de ocorrência mundial que acomete aves domésticas e silvestres, causada por Escherichia coli e resulta em perdas econômicas consideráveis devido à elevada morbidade e mortalidade das aves. O presente estudo teve o objetivo de descrever os aspectos patológicos, bacteriológicos e imuno-histoquímicos de colibacilose aviária em frangos de corte de Moçambique. Um total de 49 frangos de corte apresentaram anorexia, baixo ganho de peso, ataxia, diarreia, dispneia e morte em um curso clínico de 3 a 5 dias. As aves eram provenientes de 5 granjas (pequenas, média e grandes), com sistema de criação manual e automático, com rebanhos que variavam de 100 a 20.000 aves. À necropsia, todas as aves exibiam condição corporal ruim a caquética, além de pericárdio e cápsula de Glisson de todas aves (100%; n=49) com diferentes graus de aderência e deposição de fibrina de forma difusa acentuada. Os sacos aéreos torácicos e abdominais estavam espessados e aderidos à parede costal. Foi observado ainda hepatomegalia discreta, moderada a severa frequentemente associada com áreas multifocais puntiformes brancacentas (100%; 49/49), e esplenomegalia discreta a moderada, associado a áreas multifocais moteadas (75,5%; 37/49). Os pulmões e rins estavam aumentados e com coloração avermelhada. Histologicamente, observou-se majoritariamente serosite fibrinoheterofílica em 75,5% dos casos (37/49), caracterizadas por infiltrado inflamatório composto por heterófilos degenerados, macrófagos, linfócitos e plasmócitos, com deposição de fibrina entremeada por uma miríade de estruturas bacterianas cocobacilares. Esta lesão foi observada principalmente em pericárdio (28,6%; 14/49), pleura (18,4%; 9/49), cápsula de Glisson (10,2%; 5/49), ventrículo (10,2; 5/33) e em proventrículo (8,2%; 4/49). Áreas multifocais a coalescentes de necrose de coagulação associada a infiltrado inflamatório semelhante ao descrito foi observado principalmente no baço (28,6%; 14/49), fígado (24.5%; 12/49), e intestinos (8,2%; 4/49). Um infiltrado inflamatório semelhante também foi visualizado em pulmões (16,3%; 8/49), rins (16,3%; 8/49) e miocárdio (14,3%; 7/49), Colônias puras de E. coli foram identificadas e isoladas em 12 casos. Alguns órgãos (2 de cada granja) foram submetidos ao exame imuno-histoquímico anti-E. coli e marcação positiva foi visualizada em todos casos testados, como em fígado (3/3), coração (4/4), baço (1/1), pulmão (4/4), intestinos (4/4), bursa de Fabricius (1/1), rim (1/1), ventrículo (1/1) e proventrículo (1/1). Estes resultados demonstram que E. coli foi a causa de morte destas aves. Sendo assim, a adoção de boas medidas de biosseguridade e de manejo são indispensáveis para a prevenção e controle da ocorrência da doença nas granjas de frango de corte de Moçambique.(AU)

Genomic characterization of velogenic avian orthoavulavirus 1 isolates from poultry workers: Implications to emergence and its zoonotic potential towards public health

Objective: To carry out the genetic characterization and evolutionary analysis of three avian orthoavulavirus 1 (AOAV-1) isolates from poultry workers with respiratory symptoms. Methods: Using Illumina MiSeq, whole-genome sequencing was carried out to assess the evolutionary dynamics of three AOAV-1 isolates. A phylogenetic and comparative analysis of all coding genes was done using bioinformatics tools. Results: Phylogenetic analysis and genetic distance estimation suggested a close relationship among human- and avian-originated velogenic strains of genotype XIII, sub-genotype XIII.2.1. Several substitutions in the significant structural and biological motifs were exclusively identified in the human-originated strains. Conclusions: To our knowledge, this is the first report of a velogenic AOAV-1 isolate from natural infection of the human upper respiratory tract. Our findings highlight the evolution and zoonotic potential of velogenic AOAV-1 in a disease endemic setting.

Evaluation of beak and feather disease virus, avian polyomavirus and avian papillomavirus of captives psittacine birds in Seri Kembangan, Selangor, Malaysia

Aims@#Psittacine birds such as parrots, macaws, cockatoos, lovebirds and parakeets, are widely reared as household pets or at aviary due to their attractive features. However, the status of virus-causing diseases of psittacine species in Malaysia is fairly under-documented. Therefore, this study was aimed to detect the presence of three common avian viruses that infect psittacine birds, i.e. beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus. @*Methodology and results@#Faecal samples from twelve asymptomatic captive psittacine birds of different species were collected from an undisclosed animal garden in Serdang, Selangor, Malaysia. Briefly, the sample was homogenised and resuspended with SM buffer with the ratio 1:1 (weight of sample/g: volume of SM buffer/mL) before centrifugation at 1,000 × g for 20 min. The supernatant was collected and filtered before subjected to genomic DNA extraction using a commercialised kit. Polymerase chain reaction (PCR) technique was used to screen the V1, VP1 and L1 genes of beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus, respectively. Findings revealed that the samples were negative for BFDV and avian polyomavirus. However, positive results of 1.5 kbp PCR amplicon were detected for avian papillomavirus in four out of the 12 samples (33.33%), which was from the white-crested cockatoo, African grey parrot, yellow-collared macaw and Senegal parrot. Sequence analysis of the L1 gene from the Senegal parrot Poicephalus senegalus revealed 93% identity to a reference Psittacus erithacus timneh avian papillomavirus.@*Conclusion, significance and impact of study@#This study added to the limited prevalence data of three important avian viruses which infect captive psittacines in Seri Kembangan, Selangor, Malaysia. Avian papillomavirus, but not BFDV and avian polyomavirus, was detected in the collected captive psittacine birds. Therefore, a routine screening can be performed to monitor the health status of birds despite their asymptomatic manifestation, in order to prevent possible virus transmission.

Molecular Detection of Medically Important Candida species from Droppings of Pigeons (Columbiformes) and Captive Birds (Passeriformes and Psittaciformes)

Abstract Passeriformes and Psittaciformes birds and pigeons (Columba livia) are known to be reservoirs of microorganisms, and their stool allows fungi development. Since accumulated avian excreta can interfere with public health, this study aimed to perform a molecular screening of medically important Candida species in pigeon droppings in public places and birds raised in captivity. Excreta collected from captive birds (3 residences) and pigeons (4 districts) were inoculated on Sabouraud dextrose agar with chloramphenicol for Gram staining and subculture on Hicrome® Candida. Three DNA extraction methods were performed for comparison (commercial kit, in-house and by boiling) and PCR to screen 6 clinically important Candida species among the isolates. The correlation between phenotypic and molecular methods was calculated by kappa/K. Only 6 C. parapsilosis (20%) were identified from captive birds' feces among 30 isolates (80% not identified), while pigeons' feces harbored a greater diversity, with the 6 pathogenic species confirmed among 41 isolates: C. albicans (31.70%/13), C. krusei (14.63%/6), C. tropicalis (14.63%/6), C. parapsilosis (17.10%/7), C. glabrata (14.63%/6) and C. guilliermondii (7.31%/3); 100% correlation between tested methods (K = 1) for the first 3 species. Boiling DNA extraction method was fast and efficient to obtain viable DNA from Candida spp. for PCR. Our results indicate that pigeon droppings harbor more potentially pathogenic species than birds in residential captivity, which probably have non-albicans Candida less frequently isolated in infectious processes. The greater availability of nutrients may have contributed to a diversity of Candida spp. in feces from public environments.