The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Avian Influenza a H9N2 Viruses in Morocco, 2018-2019.

Low pathogenic H9N2 avian influenza (LPAI H9N2) is considered one of the most important diseases found in poultry (broiler, laying hens, breeding chickens, and turkeys). This infection causes considerable economic losses. The objective of this work was to monitor and assess the presence of avian influenza virus (AIV) H9N2 in eight different regions of Morocco using real-time RT-PCR, and to assess the phylogenetic and molecular evolution of the H9N2 viruses between 2016 and 2019. Field samples were collected from 108 farms suspected of being infected with LPAI H9N2 virus. Samples were analyzed using H9N2-specific real-time RT-PCR. Highly positive samples were subjected to virus isolation and seven isolates were fully sequenced. Low pathogenic H9N2 avian influenza virus was introduced in Morocco in 2016. We show that in 2018-2019, the virus was still present irrespective of vaccination status. Phylogenetic and molecular analyses showed mutations related to virulence, although our viruses were related to 2016 Moroccan viruses and grouped in the G1 lineage. Specific amino acid substitutions were identified in Moroccan H9N2 viruses that are believed to lead to increased resistance to antiviral drugs.

Protective Efficacy Evaluation of Four Inactivated Commercial Vaccines Against Low Pathogenic Avian Influenza H9N2 Virus Under Experimental Conditions in Broiler Chickens.

Avian influenza vaccines are commonly used in the poultry industry. The objective of this study was to compare, under experimental conditions, the protective efficacy of four imported commercial inactivated H9N2 vaccines (A, B, C, and D) in broiler chickens. A total of 150 one-day-old chicks were divided into six groups: four experimental groups, each containing 30 chicks, received one of the vaccines (A, B, C, or D) delivered in a 0.3-ml dose subcutaneously at 1 day of age, whereas the control, Group T, was not vaccinated but challenged and Group E was kept unvaccinated and unchallenged. At 21 days postvaccination, Groups A, B, C, D, and T were challenged with 107 embryo infective dose 50% of A/Chicken/Morocco/01/2016 (H9N2). All chicks were observed daily for clinical signs during the 12 days postchallenge (dpc). At 5 and 12 dpc, chicks were euthanatized for necropsy examination. Blood samples were collected weekly for serologic analysis and oropharyngeal swabs were collected for virus detection by real-time RT-PCR. Respiratory signs started at 48 hr pc and maximum severity was observed on 9 dpc. Chiefly, the birds vaccinated with vaccine B showed significantly more respiratory signs than did their counterparts. Serologic analysis revealed that the sera of Groups A and D birds showed a decrease in antibody (Ab) levels up to day 26; then a slight increase of Ab level was observed until day 31, while Group B and C birds showed a stabilization of the titers from day 21 until the end of the experiment. The viral shedding rate was significantly lower in Groups C and A (40%-50% of the birds shed virus for <7 days) compared with other challenged groups (60%-75% of the birds shed virus for ≥9 days). This experiment illustrated that vaccination applied on the first day in the hatchery with the four vaccines tested did not provide an acceptable protection against H9N2 in comparison with the controls that did not receive any vaccine. However, at first glance, we might favor vaccines A and C for their ability to reduce and shorten viral shedding as compared with vaccines B and D.

Evaluación de la eficacia protectora de cuatro vacunas comerciales inactivadas contra el virus de la influenza aviar H9N2 de baja patogenicidad bajo condiciones experimentales en pollos de engorde. Las vacunas contra la influenza aviar se utilizan comúnmente en la industria avícola. El objetivo de este estudio fue comparar, en condiciones experimentales, la eficacia protectora de cuatro vacunas H9N2 inactivadas comerciales importadas (A, B, C y D) en pollos de engorde. Un total de 150 pollitos de un día se dividieron en seis grupos: cuatro grupos experimentales, cada uno con 30 pollitos, recibieron una de las vacunas (A, B, C o D) administradas en una dosis de 0.3 ml por vía subcutánea al día. de edad, mientras que el control, Grupo T, que no fue vacunado y desafiado y el Grupo E que se mantuvo sin vacunar y sin desafiar. A los 21 días después de la vacunación, los Grupos A, B, C, D y T fueron desafiados con 107 dosis infecciosas de embriones al 50% del virus A/Chicken/Marruecos/01/2016 (H9N2). Todos los pollos fueron observados diariamente para detectar signos clínicos durante los 12 días posteriores al desafío (dpc). A los cinco y 12 días después del desafío, los polluelos fueron sacrificados humanitariamente para un examen de necropsia. Se recolectaron muestras de sangre semanalmente para análisis serológicos y se recolectaron hisopos orofaríngeos para la detección de virus mediante RT-PCR en tiempo real. Los signos respiratorios comenzaron a las 48 horas después del desafío y la severidad máxima se observó a los nueve días después del desafío. Principalmente, las aves vacunadas con la vacuna B mostraron significativamente más signos respiratorios que sus contrapartes. El análisis serológico reveló que los sueros de las aves de los Grupos A y D mostraron una disminución en los niveles de anticuerpos (Ab) hasta el día 26; luego se observó un ligero aumento del nivel de anticuerpos hasta el día 31, mientras que las aves de los Grupos B y C mostraron una estabilización de los títulos desde el día 21 hasta el final del experimento. La tasa de excreción viral fue significativamente menor en los Grupos C y A (40% -50% de las aves excretaron el virus durante <7 días) en comparación con otros grupos desafiados (60% -75% de las aves excretaron el virus durante ≥9 días). Este experimento ilustró que la vacunación aplicada el primer día en la incubadora con las cuatro vacunas probadas no proporcionó una protección aceptable contra el virus H9N2 en comparación con los controles que no recibieron ninguna vacuna. Sin embargo, a primera vista, podríamos favorecer las vacunas A y C por su capacidad para reducir y acortar la diseminación viral en comparación con las vacunas B y D.