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.

Recombinant A(H6N1)-H274Y avian influenza virus with dual drug resistance does not require permissive mutations to retain the replicative fitness in vitro and in ovo.

The possible emergence of drug-resistant avian flu raises concerns over the limited effectiveness of currently approved antivirals (neuraminidase inhibitors - NAIs) in the hypothetical event of a zoonotic spillover. Our study demonstrated that the recombinant avian A(H6N1) viruses showed reduced inhibition (RI) by multiple NAI drugs following the introduction of point mutations found predominantly in the neuraminidase gene (NA) of NAI-resistant human influenza strains (E119V, R292K and H274Y; N2 numbering). Moreover, A(H6N1)-H274Y showed increased replication efficiency in vitro, and a fitness advantage over wild-type (WT) when co-inoculated into embryonated hen's eggs. The results presented in our study together with the zoonotic potential of the A(H6N1) virus as evidenced by the human infection from 2013, highlight the need for enhanced monitoring of NAI resistance-associated signatures in circulating LPAI (low pathogenic avian influenza) globally.

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.

Alteration of the Chicken Upper Respiratory Microbiota, Following H9N2 Avian Influenza Virus Infection.

Several studies have highlighted the importance of the gut microbiota in developing immunity against viral infections in chickens. We have previously shown that H9N2 avian influenza A virus (AIV) infection retards the diversity of the natural colon-associated microbiota, which may further influence chicken health following recovery from infection. The effects of influenza infection on the upper respiratory tract (URT) microbiota are largely unknown. Here, we showed that H9N2 AIV infection lowers alpha diversity indices in the acute phase of infection in the URT, largely due to the family Lactobacillaceae being highly enriched during this time in the respiratory microbiota. Interestingly, microbiota diversity did not return to levels similar to control chickens in the recovery phase after viral shedding had ceased. Beta diversity followed a similar trend following the challenge. Lactobacillus associate statistically with the disturbed microbiota of infected chickens at the acute and recovery phases of infection. Additionally, we studied age-related changes in the respiratory microbiota during maturation in chickens. From 7 to 28 days of age, species richness and evenness were observed to advance over time as the microbial composition evolved. Maintaining microbiota homeostasis might be considered as a potential therapeutic target to prevent or aid recovery from H9N2 AIV infection.

Newcastle Disease Genotype VII Prevalence in Poultry and Wild Birds in Egypt.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019-2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70-99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

A random priming amplification method for whole genome sequencing of SARS-CoV-2 virus.

BACKGROUND: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. METHODS: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. RESULTS: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. CONCLUSIONS: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

A Universal, Single-Primer Amplification Protocol to Perform Whole-Genome Sequencing of Segmented dsRNA Avian Orthoreoviruses.

The Reoviridae family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus-single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5' and 3' ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses.

Protocolo universal de amplificación con un iniciador único para realizar la secuenciación del genoma completo de orthoreovirus aviares con ARN de doble cadena y segmentados La familia Reoviridae representa la familia más grande de virus de ARN de doble cadena y se han aislado miembros de una amplia variedad de mamíferos, aves, reptiles, peces, insectos y plantas. El género Orthoreovirus, uno de los 15 géneros reconocidos en la familia Reoviridae, pueden infectar a humanos y a casi todos los mamíferos y aves. La caracterización genómica de los reovirus no se ha adoptado a gran escala debido a la complejidad de obtener secuencias para los 10 segmentos. En este estudio, desarrollamos un método práctico y eficiente para enriquecer las lecturas de secuenciación de reovirus a partir de aislamientos que permite la recuperación del genoma completo utilizando un método de amplificación con un iniciador único junto con la secuenciación de próxima generación. Nos referimos a este protocolo como amplificación de un solo iniciador de reovirus (R-SPA). Estos resultados demuestran que la mayoría de los genes están cubiertos con al menos 500 lecturas por espacio base. Además, el método R-SPA cubre los extremos 5' y 3' de cada gene de reovirus. En resumen, este estudio presenta un protocolo de amplificación rápido y universal que produce suficiente ADN complementario de doble cadena y facilita y acelera la secuenciación del genoma completo de los reovirus.

Diverse Single-Stranded DNA Viruses Identified in Chicken Buccal Swabs.

High-throughput sequencing approaches offer the possibility to better understand the complex microbial communities associated with animals. Viral metagenomics has facilitated the discovery and identification of many known and unknown viruses that inhabit mucosal surfaces of the body and has extended our knowledge related to virus diversity. We used metagenomics sequencing of chicken buccal swab samples and identified various small DNA viruses with circular genome organization. Out of 134 putative circular viral-like circular genome sequences, 70 are cressdnaviruses and 26 are microviruses, whilst the remaining 38 most probably represent sub-genomic molecules. The cressdnaviruses found in this study belong to the Circoviridae, Genomoviridae and Smacoviridae families as well as previously described CRESS1 and naryavirus groups. Among these, genomoviruses and smacoviruses were the most prevalent across the samples. Interestingly, we also identified 26 bacteriophages that belong to the Microviridae family, whose members are known to infect enterobacteria.

Low pathogenic avian influenza virus infection retards colon microbiota diversification in two different chicken lines.

BACKGROUND: A commensal microbiota regulates and is in turn regulated by viruses during host infection which can influence virus infectivity. In this study, analysis of colon microbiota population changes following a low pathogenicity avian influenza virus (AIV) of the H9N2 subtype infection of two different chicken breeds was conducted. METHODS: Colon samples were taken from control and infected groups at various timepoints post infection. 16S rRNA sequencing on an Illumina MiSeq platform was performed on the samples and the data mapped to operational taxonomic units of bacterial using a QIIME based pipeline. Microbial community structure was then analysed in each sample by number of observed species and phylogenetic diversity of the population. RESULTS: We found reduced microbiota alpha diversity in the acute period of AIV infection (day 2-3) in both Rhode Island Red and VALO chicken lines. From day 4 post infection a gradual increase in diversity of the colon microbiota was observed, but the diversity did not reach the same level as in uninfected chickens by day 10 post infection, suggesting that AIV infection retards the natural accumulation of colon microbiota diversity, which may further influence chicken health following recovery from infection. Beta diversity analysis indicated a bacterial species diversity difference between the chicken lines during and following acute influenza infection but at phylum and bacterial order level the colon microbiota dysbiosis was similar in the two different chicken breeds. CONCLUSION: Our data suggest that H9N2 influenza A virus impacts the chicken colon microbiota in a predictable way that could be targeted via intervention to protect or mitigate disease.

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.

Efficacy of Two Licensed Avian Influenza H5 Vaccines Against Challenge with a 2015 U.S. H5N2 clade 2.3.4.4 Highly Pathogenic Avian Influenza Virus in Domestic Ducks.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses from the H5 goose/Guangdong lineage caused a major outbreak in poultry in the United States in 2015. Although the outbreak was controlled, vaccines were considered as an alternative control method, and new vaccines were approved and purchased by the U.S. Department of Agriculture National Veterinary Stockpile for emergency use. In this study, we evaluated the efficacy of two of these vaccines in protecting Pekin ducks (Anas platyrhynchos var. domestica) against challenge with a H5N2 HPAI poultry isolate. A recombinant alphavirus-based vaccine and an inactivated adjuvanted reverse genetics vaccine, both expressing the hemagglutinin gene of a U.S. H5 clade 2.3.4.4 isolate (A/Gyrfalcon/Washington/41088-6/2014 H5N8), were used to immunize the ducks. The vaccines were given either as single vaccination at 2 days of age or in a prime-boost strategy at 2 and 15 days of age. At 32 days of age, all ducks were challenged with A/turkey/Minnesota/12582/15 H5N2 HPAI virus clade 2.3.4.4. All ducks from the nonvaccinated challenge control group became infected and shed virus; one duck in this group presented mild ataxia, and a second duck died. No mortality or clinical signs were observed in vaccinated and challenged ducks, with the exception of one duck presenting with mild ataxia. Both vaccines, regardless of the vaccination strategy used, were immunogenic in ducks and reduced or prevented virus shedding after challenge. In conclusion, good protection against H5Nx infection was achieved in ducks vaccinated with the vaccines examined, which were homologous to the challenge virus, with prime-boost strategies conferring the best protection against infection.

Eficacia de dos vacunas con licencia contra influenza aviar H5 frente a un desafío con un virus de la influenza aviar altamente patógeno H5N2 en patos domésticos de los Estados Unidos del año 2015 y del clado 2015 2.3.4.4. Los virus de la influenza aviar altamente patógena (HPAI) 2.3.4.4 del linaje H5 ganso/Guangdong causaron un brote importante en la avicultura de los Estados Unidos en el año 2015. Aunque el brote fue controlado, las vacunas se consideraron un método de control alternativo y nuevas vacunas fueron aprobadas y adquiridas por la Reserva Nacional Veterinaria del Departamento de Agricultura de los Estados Unidos para uso en caso de emergencia. En este estudio, se evaluó la eficacia de dos de estas vacunas en la protección de patos Pekin frente al desafío con un aislamiento aviar H5N2 de alta patogenicidad. Se utilizaron una vacuna recombinante basada en alfavirus y una vacuna generada por genética inversa, inactivada y con adyuvante, ambas expresando el gene de la hemaglutinina de un aislamiento H5 clado 2.3.4.4 (A/Gyrfalcon/Washington/41088-6/2014 H5N8), para inmunizar los patos. Las vacunas se administraron como vacunación única a los 2 días de edad o con un programa de primovacunación y refuerzo a los 2 y 15 días de edad. A los 32 días de edad, todos los patos fueron desafiados con el virus de alta patogenicidad A/turkey/Minnesota/12582/15 H5N2 clado 2.3.4.4. Todos los patos del grupo control no vacunado y desafiado se infectaron y excretaron al virus; un pato en este grupo presentó ataxia leve y un segundo pato murió. No se observó mortalidad o signos clínicos en patos vacunados y desafiados, con la excepción de un pato que presentó ataxia leve. Ambas vacunas, independientemente de la estrategia de vacunación utilizada, fueron inmunogénicas en patos y redujeron o evitaron la diseminación del virus después del desafío. En conclusión, se logró una buena protección contra la infección por H5N2 en los patos vacunados con las vacunas evaluadas, las cuales eran homólogas al virus de desafío y las estrategias de primovacunación y refuerzo confirieron la mejor protección contra la infección.

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.

Characterization of H9N2 avian influenza viruses from the Middle East demonstrates heterogeneity at amino acid position 226 in the hemagglutinin and potential for transmission to mammals.

Next-generation sequencing (NGS) technologies are a valuable tool to monitor changes in viral genomes and determine the genetic heterogeneity of viruses. In this study, NGS was applied to clinical poultry samples from Jordan to detect eleven H9N2 low pathogenic avian influenza viruses (LPAIV). All of the viruses tested belonged to Middle East A genetic group of G1 lineage. Deep sequencing demonstrated a high degree of heterogeneity of glutamine and leucine residues at position 226 in the hemagglutinin (HA) gene, which increases specificity to either avian or mammalian-type receptors. Moreover, additional amino acid changes in PB1, PA, M1, M2, and NS1 were identified among the viruses tested. Compared to single gene amplification, application of NGS for surveillance and characterization of H9N2 LPAIV provides a complete genetic profile of emerging isolates and better understanding of the potential of zoonotic transmissions to mammals.

Protection of commercial turkeys following inactivated or recombinant H5 vaccine application against the 2015U.S. H5N2 clade 2.3.4.4 highly pathogenic avian influenza virus.

Between December 2014 and June 2015, North America experienced the largest recorded foreign animal disease outbreak with over 47 million poultry dead or euthanized from viral exposure to a clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) epizootic. Soon after the epizootic began, the U.S. Department of Agriculture (USDA) began testing the efficacy of different vaccines as a possible future control strategy. The aim of these studies were to evaluate the efficacy three H5 vaccines to aid in control of HPAI in commercial turkeys. Three different vaccine technologies were evaluated for efficacy: 1) inactivated reverse genetic laboratory-generated virus encoding a clade 2.3.4.4 H5 hemagglutinin (HA) gene (rgH5), 2) recombinant turkey herpesvirus encoding a clade 2.2. H5 HA (rHVT-AI), and 3) recombinant replication-deficient alphavirus RNA particle vaccine encoding a clade 2.3.4.4 H5 HA (RP-H5). All vaccines tested significantly (P<0.01) increased survival rates between vaccinated and sham vaccinated groups of poults challenged with A/turkey/Minnesota/12582/2015 clade 2.3.4.4 H5N2 HPAI. The rgH5 vaccine had detectable serum hemagglutination inhibition (HI) antibody against the challenge virus, and significantly reduced the frequency and level of viral shedding from oropharyngeal and cloacal swabs at days 2 and 4 post-challenge. Vaccination with only rHVT-AI or RP-H5 was not 100% protective, and failed to significantly reduce viral shedding post-challenge. A combined prime and boost strategy with the rHVT-AI and RP-H5, or rHVT-AI and rgH5, was 100% protective against lethal H5N2 HPAI challenge. Results of these studies led to USDA conditional approval of commercially available recombinant vaccines for use in turkeys as a control measure for clade 2.3.4.4 H5 HPAI epizootics.

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses.

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combination with MiSeq platform was applied to target negative- and positive-sense single-stranded RNA viral sequences. This method allowed successful sequence assembly of full or near full length avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) viral genome. Moreover, SISPA analysis applied to unknown clinical cases of mixed viral infections produced genome assemblies comprising 98% NDV and 99% of IBV genomes. Complete or near complete virus genome sequence was obtained with titers at or above 104.5 EID50/ml (50% embryo infectious dose), and virus identification could be detected with titers at or above 103 EID50/ml. Taken together, these studies demonstrate a simple template enrichment protocol for rapid detection and accurate characterization of avian RNA viruses.

Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 U.S. H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses.

From December 2014 to June 2015, a novel H5 Eurasian A/goose/Guangdong (Gs/GD) lineage clade 2.3.4.4 high pathogenicity avian influenza (HPAI) virus caused the largest animal health emergency in US history resulting in mortality or culling of greater than 48 million poultry. The outbreak renewed interest in developing intervention strategies, including vaccines, for these newly emergent HPAI viruses. In these studies, several existing H5 vaccines or vaccine seed strains with varying genetic relatedness (85-100%) to the 2.3.4.4 HPAI viruses were evaluated for protection in poultry. Chickens received a single dose of either an inactivated whole H5 AI vaccine, or a recombinant fowl poxvirus or turkey herpesvirus-vectored vaccines with H5 AI hemagglutinin gene inserts followed by challenge with either a U.S. wild bird H5N8 (A/gyrfalcon/Washington/40188-6/2014) or H5N2 (A/northern pintail/Washington/40964/2014) clade 2.3.4.4 isolate. Results indicate that most inactivated H5 vaccines provided 100% protection from lethal effects of H5N8 or H5N2 challenge. In contrast, the recombinant live vectored vaccines only provided partial protection which ranged from 40 to 70%. Inactivated vaccine groups, in general, had lower number of birds shedding virus and at lower virus titers then the recombinant vaccine groups. Interestingly, prechallenge antibody titers using the HPAI challenge viruses as antigen in heterologous vaccine groups were typically low (≤2 log2), yet the majority of these birds survived challenge. Taken together, these studies suggest that existing vaccines when used in a single immunization strategy may not provide adequate protection in poultry against the 2.3.4.4 HPAI viruses. Updating the H5 hemagglutinin to be genetically closer to the outbreak virus and/or using a prime-boost strategy may be necessary for optimal protection.

Mycoplasma pulmonis of Rodents as a Possible Human Pathogen.

Mycoplasma pulmonis is a naturally occurring respiratory pathogen in rodents. To date, this pathogen was not isolated from humans. This study aimed to evaluate the prevalence and seropositivity to M. pulmonis in humans who have had direct contact with rats. Moreover, the prevalence of M. pulmonis in pet and laboratory rats was assessed. Overall, 131 and 235 oropharyngeal swab samples were collected from human individuals and rats, respectively. In humans, M. pulmonis was detected by PCR in 21 of 86 pet rat keepers (24.42%), 10 of 13 technicians (76.32%), and 8 of 32 (25.0%) veterinarians. In rats, M. pulmonis was identified by PCR in 86 of 122 pet rats (70.49%) and 56 of 113 (49.56%) laboratory rats. Seroprevalence in humans was examined by screening sera from 44 individuals for M. pulmonis-specific IgG using ELISA. In total, 26 out of 44 (59.09%) humans were seropositive to M. pulmonis (4 out of 9 technicians, 8 of 12 veterinarians, and 15 of 23 pet rat keepers).The high antibody titer was found in 4 individuals (2 pet rat keepers and 2 veterinarians), whereas the moderate and low antibody titers were found in 8 and 14 individuals, respectively. The high antibody titer found in humans might indicate an active infection. However, it is unknown whether the presence of M. pulmonis in humans might be associated with disease and whether the foreign Mycoplasma can survive for long in its new environment.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.

Vaccine Protection of Turkeys Against H5N1 Highly Pathogenic Avian Influenza Virus with a Recombinant Turkey Herpesvirus Expressing the Hemagglutinin Gene of Avian Influenza.

Outbreaks of H5 highly pathogenic avian influenza (HPAI) in commercial poultry are a constant threat to animal health and food supplies. While vaccination can enhance protection and reduce the spread of disease, there is considerable evidence that the level of immunity required for protection varies by subtype and virulence of field virus. In this study, the efficacy of a recombinant turkey herpesvirus (rHVT) vector vaccine expressing the hemagglutinin gene from a clade 2.2 AI virus (A/Swan/Hungary/4999/2006) was evaluated in turkeys for protection against challenge with A/Whooper Swan/Mongolia/L244/2005 H5N1 HPAI clade 2.2. One-day-old turkeys received a single vaccination and were challenged at 4 wk postvaccination with 2 × 10(6) 50% embryo infectious dose per bird. The results demonstrate that following H5N1 HPAI challenge 96% protection was observed in rHVT-AI vaccinated turkeys. The oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared with sham-vaccinated birds. From respiratory and gastrointestinal tracts, there was a greater than 6 log10 reduction in shedding in vaccinated birds as compared with the controls. This study provides support for the use of a commercially available rHVT-AI vaccine to protect turkeys against H5N1 HPAI.

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.