The pathogenesis of a 2022 North American highly pathogenic clade 2.3.4.4b H5N1 avian influenza virus in mallards (Anas platyrhynchos).

Highly pathogenic (HP) avian influenza viruses (AIVs) of the clade 2.3.4.4 goose/Guangdong/1996 H5 lineage continue to be a problem in poultry and wild birds in much of the world. The recent incursion of a H5N1 clade 2.3.4.4b HP AIV from this lineage into North America has resulted in widespread outbreaks in poultry and consistent detections of the virus across diverse families of birds and occasionally mammals. To characterize the pathobiology of this virus in mallards (Anas platyrhynchos), which are a primary reservoir of AIV, a challenge study was conducted with 2-week-old birds. The 50% bird infectious dose was determined to be < 2 log10 50% egg infectious doses (EID50) and all exposed ducks, including ducks co-housed with inoculated ducks, were infected. Infection appeared to be subclinical for 58.8% (20/34) of the ducks, one duck was lethargic, about 20% developed neurological signs and were euthanized, and 18% developed corneal opacity. The mallards shed virus by both the oral and cloacal routes within 24-48 h post-infection. Oral shedding substantially decreased by 6-7 days post-infection, but 65% of the ducks continued to shed virus cloacally through 14 days post-exposure (DPE) for the direct inoculates and 13 DPE for contact-exposed ducks. Based on the high transmissibility, high virus shed titres, and mild-to-moderate disease, mallards could serve as efficient reservoirs to amplify and disseminate recent North American clade 2.3.4.4b viruses.

Lack of Susceptibility to SARS-CoV-2 and MERS-CoV in Poultry.

We challenged chickens, turkeys, ducks, quail, and geese with severe acute respiratory syndrome coronavirus 2 or Middle East respiratory syndrome coronavirus. We observed no disease and detected no virus replication and no serum antibodies. We concluded that poultry are unlikely to serve a role in maintenance of either virus.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens.

Highly pathogenic avian influenza virus (HPAIV) has caused widespread outbreaks in poultry in the Americas. Because of the duration and extent of these outbreaks, vaccine use may be an additional tool to limit virus spread. Three vaccines were evaluated for efficacy in chickens against a current North American clade 2.3.4.4b H5 HPAIV isolate, A/turkey/Indiana/3703-003/2022 H5N1. The vaccines included: 1) a commercial inactivated reverse genetics (rg) generated H5N1 product with a clade 2.3.4.4c H5 hemagglutinin (HA) (rgH5N1); 2) a commercial alphavirus RNA particle (RP) vaccine with the TK/IN/22 HA; and 3) an in-house inactivated rg produced vaccine with the TK/IN/22 HA and a North American lineage N9 neuraminidase (NA) (SEP-22-N9). Both inactivated vaccines were produced with HA genes that were modified to be low pathogenic and with the remaining genes from the PR8 influenza strain. All vaccines provided 100% protection against mortality and morbidity and all vaccines reduced virus shed by the oropharyngeal and cloacal routes significantly compared to sham vaccinates. However, differences were observed among the vaccines in quantities of virus shed at two- and four-days post challenge (DPC). To determine if infected birds could be identified after vaccination to aid surveillance programs, serum was collected from the RP and SEP-22-N9 vaccine groups at 7, 10, and 14 DPC to detect antibody to the NA and nucleoprotein (NP) of the challenge virus by enzyme linked lectin assay (ELLA) and ELISA. As early as 7DPC ELLA detected antibody in sera from 100% of the chickens in the RP vaccinated group and 70% of the chickens in the SEP-22-N9 vaccinated group. Antibody to the NP was detected by commercial ELISA in more than 50% of the birds in the RP vaccinated group at each time point.

Pathogenicity in Chickens and Turkeys of a 2021 United States H5N1 Highly Pathogenic Avian Influenza Clade 2.3.4.4b Wild Bird Virus Compared to Two Previous H5N8 Clade 2.3.4.4 Viruses.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have infected many wild and domestic birds. We evaluated the pathobiology of an early U.S. H5N1 HPAIV (clade 2.3.4.4b, 2021) and two H5N8 HPAIVs from previous outbreaks in the U.S. (clade 2.3.4.4c, 2014) and Europe (clade 2.3.4.4b, 2016) in chickens and turkeys. Differences in clinical signs, mean death times (MDTs), and virus transmissibility were found between chickens and turkeys. The mean bird infective dose (BID50) of the 2021 H5N1 virus was approximately 2.6 log10 50% embryo infective dose (EID50) in chickens and 2.2 log10 EID50 in turkeys, and the virus transmitted to contact-exposed turkeys but not chickens. The BID50 for the 2016 H5N8 virus was also slightly different in chickens and turkeys (4.2 and 4.7 log10 EID50, respectively); however, the BID50 for the 2014 H5N8 virus was higher for chickens than turkeys (3.9 and ~0.9 log10 EID50, respectively). With all viruses, turkeys took longer to die (MDTs of 2.6-8.2 days for turkeys and 1-4 days for chickens), which increased the virus shedding period and facilitated transmission to contacts.

Avian Influenza Virus RNA Extraction.

The efficient extraction and purification of viral RNA is critical for downstream molecular applications such as the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, gene sequencing, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types: enriched (e.g., virus stocks) and non-enriched (e.g., clinical). Clinical samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the extraction of AI virus RNA from common clinical specimen types and enriched virus stocks will be presented.

Trends in embryonic and ontogenetic growth metabolisms in nonavian dinosaurs and extant birds, mammals, and crocodylians with implications for dinosaur egg incubation.

The embryonic metabolism of the saurischian dinosaur Troodon formosus and the ornithischian dinosaurs Protoceratops andrewsi and Hypacrosaurus stebingeri have been determined by using a mass growth model based on conservation of energy and found to be very similar. Embryonic and ontogenetic growth metabolisms are also evaluated for extant altricial birds, precocial birds, mammals, and crocodylians to examine for trends in the different groups of animals and to provide a context for interpreting our results for nonavian dinosaurs. This analysis reveals that the embryonic metabolisms of these nonavian dinosaurs were closer to the range observed in extant crocodylians than extant birds. The embryonic metabolisms of nonavian dinosaurs were in the range observed for extant mammals of similar masses. The measured embryonic metabolic rates for these three nonavian dinosaurs are then used to calculate the incubation times for eggs of 22 nonavian dinosaurs from both Saurischia and Ornithischia. The calculated incubation times vary from about 50 days for Archaeopteryx lithographica to about 150 days for Alamosaurus sanjuanensis.

Embryonic metabolism of the ornithischian dinosaurs Protoceratops andrewsi and Hypacrosaurus stebingeri and implications for calculations of dinosaur egg incubation times.

The embryonic metabolisms of the ornithischian dinosaurs Protoceratops andrewsi and Hypacrosaurus stebingeri have been determined and are in the range observed in extant reptiles. The average value of the measured embryonic metabolic rates for P. andrewsi and H. stebingeri are then used to calculate the incubation times for 21 dinosaurs from both Sauischia and Ornithischia using a mass growth model based on conservation of energy. The calculated incubation times vary from about 70 days for Archaeopteryx lithographica to about 180 days for Alamosaurus sanjuanensis. Such long incubation times seem unlikely, particularly for the sauropods and large theropods. Incubation times are also predicted with the assumption that the saurischian dinosaurs had embryonic metabolisms in the range observed in extant birds.

Incubation times of dinosaur eggs via embryonic metabolism.

The incubation times for the eggs of 21 dinosaurs are determined from an estimate of their embyronic metabolic rate and the mass of the hatchlings via a mass growth model based on conservation of energy. Embryos in extant birds and crocodiles are studied in order to determine the best model for embryonic metabolism and growth. These results are used to develop a theoretical model that predicts the incubation times of an egg. This model is applied to dinosaur eggs and provides a unique window into dinosaur reproduction. The dinosaurs studied come from both Saurischia and Ornithischia. The incubation times vary from about 28 days for Archaeopteryx lithographica to about 76 days for Alamosaurus sanjuanensis.

A high pressure study of the eigenvectors of the infra-red active vibrational modes of crystalline adenosine.

High-pressure infrared spectroscopy has been used to study the eigenvectors and eigenvalues of the vibrational modes of crystalline adenosine at 298 K by evaluating the logarithmic derivative of the vibrational frequency with respect to pressure: [Formula: see text]. Crystalline samples of molecular materials such as adenosine have vibrational modes that are localized within a molecular unit ("internal" modes) as well as modes in which the molecular units vibrate against each other ("external" modes). The value of the logarithmic derivative is that it is a diagnostic probe of the nature of the eigenvector of these vibrational modes. Stretching modes, which are predominantly internal to the molecule, have low logarithmic derivatives while external modes have higher logarithmic derivatives. Particular attention is paid to modes in the 800-1000 cm(-1) range since modes in that region of the vibrational spectrum are found to be sensitive to the conformation of double-helical DNA. Since the sugar pucker is different for the various conformations of DNA, this fact suggests that these modes involve the motion of atoms in the sugar group. The vibrations of the hydrogen atoms are also of interest to study since the vibrational frequency of hydrogen atoms involved in hydrogen bonds has a negative pressure derivative. Such behavior clearly shows which hydrogen atoms are involved in hydrogen bonding.

Metabolism of dinosaurs as determined from their growth.

A model based on cellular properties is used to analyze the mass growth curves of 20 dinosaurs. This analysis yields the first measurement of the average cellular metabolism of dinosaurs. The organismal metabolism is also determined. The cellular metabolism of dinosaurs is found to decrease with mass at a slower rate than is observed in extant animals. The organismal metabolism increases with the mass of the dinosaur. These results come from both the Saurischia and Ornithischia branches of Dinosauria, suggesting that the observed metabolic features were common to all dinosaurs. The results from dinosaurs are compared to data from extant placental and marsupial mammals, a monotreme, and altricial and precocial birds, reptiles, and fish. Dinosaurs had cellular and organismal metabolisms in the range observed in extant mesotherms.

A study of the eigenvectors of the vibrational modes in crystalline cytidine via high-pressure Raman spectroscopy.

Raman spectroscopy has been used to study the eigenvectors and eigenvalues of the vibrational modes of crystalline cytidine at 295 K and high pressures by evaluating the logarithmic derivative of the vibrational frequency ω with respect to pressure P: [Formula: see text]. Crystalline samples of molecular materials have strong intramolecular bonds and weak intermolecular bonds. This hierarchy of bonding strengths causes the vibrational optical modes localized within a molecular unit ("internal" modes) to be relatively high in frequency while the modes in which the molecular units vibrate against each other ("external" modes) have relatively low frequencies. The value of the logarithmic derivative is a useful diagnostic probe of the nature of the eigenvector of the vibrational modes because stretching modes (which are predominantly internal to the molecule) have low logarithmic derivatives while external modes have higher logarithmic derivatives. In crystalline cytidine, the modes at 85.8, 101.4, and 110.6 cm(-1) are external in which the molecules of the unit cell vibrate against each other in either translational or librational motions (or some linear combination thereof). All of the modes above 320 cm(-1) are predominantly internal stretching modes. The remaining modes below 320 cm(-1) include external modes and internal modes, mostly involving either torsional or bending motions of groups of atoms within a molecule.

Avian influenza virus RNA extraction.

The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types; enriched (e.g. virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter two well established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented.

A study of the eigenvectors of the low-frequency vibrational modes in crystalline adenosine via high pressure Raman spectroscopy.

High-pressure Raman spectroscopy has been used to study the eigenvectors and eigenvalues of the vibrational modes of crystalline adenosine at 295 K by evaluating the logarithmic derivative of the vibrational frequency with respect to pressure: [Formula: see text]. Crystalline samples of molecular materials such as adenosine will have vibrational modes that are localized within a molecular unit ("internal" modes) as well as modes in which the molecular units vibrate against each other ("external" modes). The value of the logarithmic derivative is found to be a diagnostic probe of the nature of the eigenvector of the vibrational modes. Stretching modes which are predominantly internal to the molecule have low logarithmic derivatives while external modes have higher logarithmic derivatives. Particular interest is paid to the low-frequency (≤150 cm(-1)) modes. Based on the pressure dependence of its logarithmic derivative, a mode near 49 cm(-1) is identified as internal mode. The other modes below 400 cm(-1) have pressure dependences of their logarithmic derivatives consistent with being either (1) modes which are mainly external, meaning that the molecules of the unit cell vibrate against each other in translational or librational motions (or linear combinations thereof), or (2) torsional or bending modes involving a large number of atoms, mainly within a molecule. The modes above 400 cm(-1) all have pressure dependences of their logarithmic derivatives consistent with being mainly internal modes.

A Raman scattering study of the interactions of DNA with its water of hydration.

Raman spectroscopy is used to probe the nature of the hydrogen bonds which hold the water of hydration to DNA. The ∼ 3450 cm(-1) molecular O-H stretching mode shows that the first six water molecules per base pair of the primary hydration shell are very strongly bound to the DNA. The observed shift in the peak position of this mode permits a determination of the length of the hydrogen bonds for these water molecules. These hydrogen bonds appear to be about 0.3 Šshorter than the hydrogen bonds in bulk water. The linewidth of this mode shows no significant changes above water contents of about 15 water molecules per base pair. This technique of using a vibrational spectroscopy to obtain structural information about the hydration shells of DNA could be used to study the hydration shells of other biomolecules.

Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery.

Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency.