Genetic insertion of mouse Myxovirus-resistance gene 1 increases innate resistance against both high and low pathogenic avian influenza virus by significantly decreasing replication in chicken DF1 cell line.

Avian influenza virus (AIV) is a constant threat to animal health with recent global outbreaks resulting in the death of hundreds of millions of birds with spillover into mammals. Myxovirus-resistance (Mx) proteins are key mediators of the antiviral response that block virus replication. Mouse (Mu) Mx (Mx1) is a strong antiviral protein that interacts with the viral nucleoprotein to inhibit polymerase function. The ability of avian Mx1 to inhibit AIV is unclear. In these studies, Mu Mx1 was stably introduced into chicken DF1 cells to enhance the immune response against AIV. Following infection, titers of AIV were significantly decreased in cells expressing Mu Mx1. In addition, considerably less cytopathic effect (CPE) and matrix protein staining was observed in gene-edited cells expressing Mu Mx1, suggesting Mu Mx1 is broadly effective against multiple AIV subtypes. This work provides foundational studies for use of gene-editing to enhance innate disease resistance against AIV.

Efficacy of recombinant H5 vaccines delivered in ovo or day of age in commercial broilers against the 2015 U.S. H5N2 clade 2.3.4.4c highly pathogenic avian Influenza virus.

BACKGROUND: Avian influenza is a highly contagious, agriculturally relevant disease that can severely affect the poultry industry and food supply. Eurasian-origin H5Nx highly pathogenic avian influenza viruses (HPAIV) (clade 2.3.4.4) have been circulating globally in wild birds with spill over into commercial poultry operations. The negative impact to commercial poultry renewed interest in the development of vaccines against these viruses to control outbreaks in the U.S. METHODS: The efficacy of three recombinant H5 vaccines delivered in ovo or day of age were evaluated in commercial broilers challenged with the 2015 U.S. H5N2 clade 2.3.4.4c HPAIV. The recombinant vaccines included an alphavirus RNA particle vaccine (RP-H5), an inactivated reverse genetics-derived (RG-H5) and recombinant HVT vaccine (rHVT-AI) expressing H5 hemagglutinin (HA) genes. In the first experiment, in ovo vaccination with RP-H5 or rHVT-AI was tested against HPAI challenge at 3 or 6 weeks of age. In a second experiment, broilers were vaccinated at 1 day of age with a dose of either 107 or 108 RP-H5, or RG-H5 (512 HA units (HAU) per dose). RESULTS: In experiment one, the RP-H5 provided no protection following in ovo application, and shedding titers were similar to sham vaccinated birds. However, when the RP-H5 was delivered in ovo with a boost at 3 weeks, 95% protection was demonstrated at 6 weeks of age. The rHVT-AI vaccine demonstrated 95 and 100% protection at 3 and 6 weeks of age, respectively, of challenged broilers with reduced virus shedding compared to sham vaccinated birds. Finally, when the RP-H5 and rHVT vaccines were co-administered at one day of age, 95% protection was demonstrated with challenge at either 3 or 6 weeks age. In the second experiment, the highest protection (92%) was observed in the 108 RP-H5 vaccinated group. Significant reductions (p < 0.05) in virus shedding were observed in groups of vaccinated birds that were protected from challenge. The RG-H5 provided 62% protection from challenge. In all groups of surviving birds, antibody titers increased following challenge. CONCLUSIONS: Overall, these results demonstrated several strategies that could be considered to protected broiler chickens during a H5 HPAI challenge.

Cell-Adapted Mutations and Antigenic Diversity of Influenza B Viruses in Missouri, 2019-2020 Season.

Influenza B viruses (IBVs) are causing an increasing burden of morbidity and mortality, yet the prevalence of culture-adapted mutations in human seasonal IBVs are unclear. We collected 368 clinical samples from patients with influenza-like illness in Missouri during the 2019-2020 influenza season and recovered 146 influenza isolates including 38 IBV isolates. Of MDCK-CCL34, MDCK-Siat1, and humanized MDCK (hCK), hCK showed the highest virus recovery efficiency. All Missourian IBVs belonged to the Victoria V1A.3 lineage, all of which contained a three-amino acid deletion on the HA protein and were antigenically distant from the Victoria lineage IBV vaccine strain used during that season. By comparing genomic sequences of these IBVs in 31 paired samples, eight cell-adapted nonsynonymous mutations were identified, with the majority in the RNA polymerase. Analyses of IBV clinical sample-isolate pairs from public databases further showed that cell- and egg-adapted mutations occurred more widely in viral proteins, including the receptor and antibody binding sites on HA. Our study suggests that hCK is an effective platform for IBV isolation and that culture-adapted mutations may occur during IBV isolation. As culture-adapted mutations may affect subsequent virus studies and vaccine development, the knowledge from this study may help optimize strategies for influenza surveillance, vaccine strain selection, and vaccine development.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

SARS-CoV-2 show no infectivity at later stages in a prolonged COVID-19 patient despite positivity in RNA testing.

Inpatient coronavirus disease 2019 (COVID-19) cases present enormous costs to patients and health systems in the United States. Many hospitalized patients may continue testing COVID-19 positive even after the resolution of symptoms. Thus, a pressing concern for clinicians is the safety of discharging these asymptomatic patients if they have any remaining infectivity. This case report explores the viral viability in a patient with persistent COVID-19 over the course of a 2-month hospitalization. Positive nasopharyngeal swab samples were collected and isolated in the laboratory and analyzed by quantitative reverse-transcription polymerase chain reactions (qRT-PCR), and serology was tested for neutralizing antibodies throughout the hospitalization period. The patient experienced waning symptoms by hospital day 40 and had no viable virus growth by hospital day 41, suggesting no risk of infectivity, despite positive RT-PCR results which prolonged his hospital stay. Notably, this case showed infectivity for at least 24 days after disease onset, which is longer than the discontinuation of transmission-based precautions recommended by the Center for Disease Control and Prevention. Thus, our findings suggest that the timeline for discontinuing transmission-based precautions may need to be extended for patients with severe and prolonged COVID-19 disease. Additional large-scale studies are needed to draw definitive conclusions on the appropriate clinical management for these patients. ​.

Techniques for the Measurement of Cell Mediated Immune Responses to Avian Influenza Virus.

Cellular immune responses, through both T and B cells, are critical to understanding the role and regulation of lymphocytes following viral infection, as well as defining responses to vaccination. T cells play a critical role in adaptive immunity, including pathogen elimination through the engagement of CD4 and CD8 receptors, which trigger signaling mechanisms. B cells contribute to generating antibodies following exposure to foreign pathogens through interactions with CD4+ lymphocytes. While these different cell types have distinctly different modes of action in terms of contributions to protection (cytotoxic versus antibody mediated), they account for the majority of adaptive immunity induced following infection or vaccination. While the ability to measure cell-mediated immunity (CMI) has steadily improved, there is much to learn with regard to their contribution to the protection of birds against diseases induced by avian influenza virus. The rapidly increasing knowledge of genomic avian sequences, along with the increasing availability of monoclonal antibodies detecting avian cell-associated antigen markers, has made techniques to measure CMI more specific and informative for researchers.

The Contribution of Eimeria Coinfection and Intestinal Inflammation to Cecal Colonization and Systemic Spread of Salmonella Typhimurium Deficient in Tetrathionate Reductase or Type III Secretion Systems Salmonella Pathogenicity Island 1 or 2.

Intestinal inflammation may provide a growth advantage for Salmonella and enhance its systemic spread in chickens. Salmonella triggers intestinal inflammation in the host by using type III secretion systems (T3SS) and produces the inflammatory end product tetrathionate. In mice, tetrathionate respiration confers a growth advantage for Salmonella Typhimurium over the competitive microbiome in the inflamed intestine. Coccidia also promote intestinal inflammation and enhance Salmonella intestinal growth and systemic spread in chickens. The objective of this study was to evaluate the contribution of inflammation, induced by Eimeria spp. or Salmonella Typhimurium, to Salmonella colonization and dissemination in chickens. In addition, the fitness costs associated with defects in tetrathionate reductase and T3SS associated with Salmonella Pathogenicity Island 1 or 2 (SPI-1 or SPI-2) were evaluated in in vivo competition experiments with wild-type Salmonella strain, with or without Eimeria coinfection. One-day-old specific-pathogen-free chickens were orally inoculated with a sham inoculum or with 4 × 102Eimeria oocysts cocktail of Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria mitis. At 6 days of age, birds were orally administered a 1:1 ratio of Salmonella Typhimurium wild-type and mutant deficient in tetrathionate reductase, SPI-1, or SPI-2 (108 colony forming units/bird). Ceca, livers, and drumsticks were collected at 3, 7, 14, and 42 days after Salmonella infection, for bacteriology. Intestinal inflammation was scored by histology. Significantly higher intestinal inflammation was observed in challenge groups compared with the control. However, there were no significant differences in intestinal inflammation scores between groups coinfected with both Eimeria spp. and Salmonella Typhimurium and birds infected with Salmonella alone, and Eimeria coinfection did not increase Salmonella prevalence or abundance. Contrary to mouse studies, tetrathionate reductase did not enhance Salmonella Typhimurium cecal colonization or systemic spread in chickens. SPI-1 and SPI-2 played a significant role in Salmonella dissemination and cecal colonization in chickens, respectively.

Contribución de la coinfección por Eimeria y de la inflamación intestinal a la colonización cecal y a la propagación sistémica de Salmonella Typhimurium deficiente en tetrationato reductasa o de sistemas de secreción de tipo III de islas de patogenicidad 1 o 2 de Salmonella. La inflamación intestinal puede proporcionar una ventaja para el crecimiento de Salmonella y aumentar su propagación sistémica en pollos. Salmonella desencadena la inflamación intestinal en el huésped mediante el uso de sistemas de secreción tipo III (T3SS) y produce el producto final inflamatorio, tetrationato. En ratones, la respiración con tetrationato confiere una ventaja de crecimiento para Salmonella Typhimurium sobre el microbioma competitivo en el intestino inflamado. Coccidia también promueve la inflamación intestinal y mejora el crecimiento intestinal de Salmonella y la propagación sistémica en pollos. El objetivo de este estudio fue evaluar la contribución de la inflamación, inducida por Eimeria spp. o Salmonella Typhimurium, en la colonización y diseminación de Salmonella en pollos. Además, se evaluaron los costos de aptitud asociados con defectos en la tetrationato reductasa y T3SS asociados con las islas de patogenicidad 1 o 2 de Salmonella (SPI-1 o SPI-2) mediante experimentos de competencia in vivo con cepas de Salmonella de tipo silvestre, con o sin coinfección con Eimeria. Pollos libres de patógenos específicos de un día de edad se inocularon por vía oral con un inóculo falso o con 4 × 102 de un coctel de ooquistes de Eimeria que incluyó Eimeria tenella, Eimeria acervulina, Eimeria maxima y Eimeria mitis. A los seis días de edad, se les administró a las aves administró por vía oral una proporción 1: 1 de Salmonella Typhimurium de tipo silvestre o tipo mutante que es deficiente de tetrationato reductasa, SPI-1 o SPI-2 (108 unidades formadoras de colonias/ave). Se recolectaron ciegos, hígados y pernas a los tres, siete, catorce y 42 días después de la infección por Salmonella, para bacteriología. La inflamación intestinal se calificó por histología. Se observó inflamación intestinal significativamente mayor en los grupos de desafío en comparación con el control. Sin embargo, no hubo diferencias significativas en las puntuaciones de inflamación intestinal entre los grupos coinfectados con Eimeria spp. y Salmonella Typhimurium y las aves infectadas con Salmonella por si sola y la coinfección con Eimeria no aumentó la prevalencia o abundancia de Salmonella. A diferencia de los estudios en ratones, la tetrationato reductasa no mejoró la colonización cecal de Salmonella Typhimurium o la diseminación sistémica en pollos. Las islas de patogenicidad SPI-1 y SPI-2 jugaron un papel importante en la diseminación de Salmonella y en la colonización cecal en pollos, respectivamente.

Are Microneutralization and Hemagglutination Inhibition Assays Comparable? Serological Results from Influenza Experimentally Infected Mallard Ducks.

The hemagglutination inhibition (HI) assay is commonly used to assess the humoral immune response against influenza A viruses (IAV). However, the microneutralization (MN) assay has been reported to have higher sensitivity when testing sera from humans and other species. Our objective was to determine the agreement between MN and HI assays and compare the proportion of positive samples detected by both methods in sera of mallards primary infected with the A/mallard/MN/Sg-000169/ 2007 (H3N8) virus and subsequently inoculated with homosubtypic or heterosubtypic IAV. Overall, we found poor to fair agreement (prevalence-adjusted bias-adjusted kappa [PABAK], 0.03-0.35) between MN and HI assays in serum samples collected 2 weeks after H3N8 inoculation; the observed agreement increased to moderate or substantial in samples collected 4 to 5 weeks postinoculation (WPI) (PABAK, 0.52-0.75). The MN assay detected a higher proportion of positive samples compared with HI assays in serum samples collected 2 WPI (P = 0.01). This difference was not observed in samples collected 4 WPI. Also, a boosting effect in MN and HI titers was observed when birds were subsequently inoculated with IAV within the same H3 clade. This effect was not observed when birds were challenged with viruses that belong to a different HA clade. In summary, the agreement between assays varies depending on the postinfection sample collection time point and the similarity between the antigens used for the assays. Additionally, subsequent exposure of ducks to homosubtypic or heterosubtypic strains might affect the observed agreement.

¿Los ensayos de microneutralización e inhibición de la hemaglutinación son comparables? Resultados serológicos de patos de collar infectados experimentalmente con influenza. La prueba de inhibición de la hemaglutinación se usa rutinariamente para evaluar la respuesta inmune humoral contra los virus de influenza aviar, sin embargo, se ha reportado que la prueba de microneutralización tiene una mayor sensibilidad cuando se evalúan muestras de suero de humanos u otras especies. Este estudio tuvo como objetivo determinar la concordancia entre las pruebas de microneutralización e inhibición de la hemaglutinación en suero de patos de collar que fueron desafiados con el virus A/ mallard/MN/Sg-000169/2007(H3N8) y re-inoculados con virus de influenza aviar homosubtípicos o heterosubtípicos. Además, se comparó la proporción de muestras positivas detectadas por ambos métodos. En general, se observó un nivel de concordancia pobre a razonable (PABAK = 0.03 - 0.35) entre las pruebas de microneutralización e inhibición de la hemaglutinación en muestras de suero recolectadas dos semanas post-inoculación del virus H3N8. La concordancia se incrementó a moderada o sustancial en muestras recolectadas cuatro o cinco semanas después de la inoculación (PABAK = 0.52 - 0.75). Una mayor proporción de muestras recolectadas a las dos semanas después de la inoculación fueron positivas por microneutralización en comparación con inhibición de la hemaglutinación (P = 0.01), estas diferencias no fueron observadas con las muestras recolectadas a las cuatro semanas después de la inoculación. Adicionalmente, se observó un incremento en los títulos de anticuerpos cuando las aves fueron re-inoculadas con virus de influenza aviar pertenecientes al mismo clado H3 de la hemaglutinina. Este efecto no fue observado en los patos re-inoculados con virus de influenza aviar pertenecientes a un clado distinto. En resumen, la concordancia entre los ensayos varía según el momento de recolección de la muestra y la similitud entre los antígenos utilizados para los ensayos. Además, la re-inoculación de patos con una cepa homosubtípica or heterosubtípica podría afectar el nivel de concordancia observada.

Heterosubtypic immunity increases infectious dose required to infect Mallard ducks with Influenza A virus.

Previous field and experimental studies have demonstrated that heterosubtypic immunity (HSI) is a potential driver of Influenza A virus (IAV) prevalence and subtype diversity in mallards. Prior infection with IAV can reduce viral shedding during subsequent reinfection with IAV that have genetically related hemagglutinins (HA). In this experiment, we evaluated the effect of HSI conferred by an H3N8 IAV infection against increasing challenge doses of closely (H4N6) and distantly (H6N2) related IAV subtypes in mallards. Two groups of thirty 1-month-old mallards each, were inoculated with 105.9 50% embryo infectious doses (EID50) of an H3N8 virus or a mock-inoculum. One month later, groups of five birds each were challenged with increasing doses of H4N6 or H6N2 virus; age-matched, single infection control ducks were included for all challenges. Results demonstrate that naïve birds were infected after inoculation with 103 and 104 EID50 doses of the H4N6 or H6N2 virus, but not with 102 EID50 doses of either IAV. In contrast, with birds previously infected with H3N8 IAV, only one duck challenged with 104 EID50 of H4N6 IAV was shedding viral RNA at 2 days post-inoculation, and with H6N2 IAV, only birds challenged with the 104 EID50 dose were positive to virus isolation. Viral shedding in ducks infected with H6N2 IAV was reduced on days 2 and 3 post-inoculation compared to control birds. To explain the differences in the dose necessary to produce infection among H3-primed ducks challenged with H4N6 or H6N2 IAV, we mapped the amino acid sequence changes between H3 and H4 or H6 HA on predicted three-dimensional structures. Most of the sequence differences occurred between H3 and H6 at antigenic sites A, B, and D of the HA1 region. These findings demonstrate that the infectious dose necessary to infect mallards with IAV can increase as a result of HSI and that this effect is most pronounced when the HA of the viruses are genetically related.

Adaptive Heterosubtypic Immunity to Low Pathogenic Avian Influenza Viruses in Experimentally Infected Mallards.

Mallards are widely recognized as reservoirs for Influenza A viruses (IAV); however, host factors that might prompt seasonality and trends in subtype diversity of IAV such as adaptive heterosubtypic immunity (HSI) are not well understood. To investigate this, we inoculated mallards with a prevailing H3N8 low pathogenic avian influenza virus (LPAIV) subtype in waterfowl to determine if prior infection with this virus would be protective against heterosubtypic infections with the H4N6, H10N7 and H14N5 LPAIV subtypes after one, two and three months, respectively. Also, we investigated the effect of cumulative immunity after sequential inoculation of mallards with these viruses in one-month intervals. Humoral immunity was assessed by microneutralization assays using a subset of representative LPAIV subtypes as antigens. Our results indicate that prior inoculation with the H3N8 virus confers partial protective immunity against subsequent heterosubtypic infections with the robustness of HSI related to the phylogenetic similarity of the HA protein of the strains used. Furthermore, induced HSI was boosted and followed by repeated exposure to more than one LPAIV subtype. Our findings provide further information on the contributions of HSI and its role in the dynamics of IAV subtype diversity in mallards.

The Genetic Diversity of Influenza A Viruses in Wild Birds in Peru.

Our understanding of the global ecology of avian influenza A viruses (AIVs) is impeded by historically low levels of viral surveillance in Latin America. Through sampling and whole-genome sequencing of 31 AIVs from wild birds in Peru, we identified 10 HA subtypes (H1-H4, H6-H7, H10-H13) and 8 NA subtypes (N1-N3, N5-N9). The majority of Peruvian AIVs were closely related to AIVs found in North America. However, unusual reassortants, including a H13 virus containing a PA segment related to extremely divergent Argentinian viruses, suggest that substantial AIV diversity circulates undetected throughout South America.

Characterization and Sequencing of a Genotype XII Newcastle Disease Virus Isolated from a Peacock (Pavo cristatus) in Peru.

Here, we report the first complete sequence and biological characterization of a Newcastle disease virus (NDV) isolated from a peacock in South America (NDV/peacock/Peru/2011). This isolate, classified as genotype XII in class II, highlights the need for increased surveillance of noncommercial avian species.