Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in the skin was 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

Comparative Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Variants in the Ferret Model.

The continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans necessitates evaluation of variants for enhanced virulence and transmission. We used the ferret model to perform a comparative analysis of four SARS-CoV-2 strains, including an early pandemic isolate from the United States (WA1), and representatives of the Alpha, Beta, and Delta lineages. While Beta virus was not capable of pronounced replication in ferrets, WA1, Alpha, and Delta viruses productively replicated in the ferret upper respiratory tract, despite causing only mild disease with no overt histopathological changes. Strain-specific transmissibility was observed; WA1 and Delta viruses transmitted in a direct contact setting, whereas Delta virus was also capable of limited airborne transmission. Viral RNA was shed in exhaled air particles from all inoculated animals but was highest for Delta virus. Prior infection with SARS-CoV-2 offered varied protection against reinfection with either homologous or heterologous variants. Notable genomic variants in the spike protein were most frequently detected following WA1 and Delta virus infection. IMPORTANCE Continued surveillance and risk assessment of emerging SARS-CoV-2 variants are critical for pandemic response and preparedness. As such, in vivo evaluations are indispensable for early detection of variants with enhanced virulence and transmission. Here, we used the ferret model to compare the pathogenicity and transmissibility of an original SARS-CoV-2 isolate (USA-WA1/2020 [WA1]) to those of a panel of Alpha, Beta, and Delta variants, as well as to evaluate protection from homologous and heterologous reinfection. We observed strain-specific differences in replication kinetics in the ferret respiratory tract and virus load emitted into the air, revealing enhanced transmissibility of the Delta virus relative to previously detected strains. Prior infection with SARS-CoV-2 provided varied levels of protection from reinfection, with the Beta strain eliciting the lowest level of protection. Overall, we found that ferrets represent a useful model for comparative assessments of SARS-CoV-2 infection, transmission, and reinfection.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

First human infection of avian influenza A(H5N6) virus reported in Lao People's Democratic Republic, February-March 2021.

In March 2021, Lao People's Democratic Republic (Laos) reported an avian influenza A(H5N6) virus infection in a 5-year-old child identified through sentinel surveillance. This was the first human A(H5N6) infection reported outside of China. A multidisciplinary investigation undertook contact tracing and enhanced human and animal surveillance in surrounding villages and live bird markets. Seven Muscovy ducks tested positive for highly pathogenic avian influenza A(H5N6) viruses. Sequenced viruses belonged to clade 2.3.4.4h and were closely related to viruses detected in poultry in Vietnam and to previous viruses detected in Laos. Surveillance and coordinated outbreak response remain essential to global health security.

Modulation of folliculogenesis in adult laying chickens by bisphenol A and bisphenol S: Perspectives on ovarian morphology and gene expression.

Both bisphenol A (BPA) and its analog bisphenol S (BPS) are industrial chemicals that have been used to make certain plastic products applied in chicken farms, including food and water containers. They are endocrine disrupting chemicals (EDCs) with xenoestrogenic activities and affect reproductive success in many ways. It was hypothesized that BPA and BPS could adversely affect the folliculogenesis in chickens due to their disruption of the estrogen responses, using either genomic or non-genomic mechanisms. This study investigated the deleterious effects of BPA and BPS on the ovaries when adult layer chickens were orally treated with these EDCs at 50 µg/kg body weight, the reference dose for chronic oral exposure of BPA established by the U.S. EPA. The chickens in both BPA and BPS-treated groups showed a decreased number of the preovulatory follicles. BPA-treated chickens showed a significant decrease in the diameter of F1. Additionally, both BPA and BPS treatments increased the infiltrations of lymphocytes and plasma cells in ovaries. Moreover, it was found that the ovaries of BPS-treated chickens weighed the most among the groups. RNA sequencing and subsequent pathway enrichment analysis of differentially expressed genes revealed that both BPA- and BPS-treatment groups showed significant changes in gene expression and pathways related to reproduction, immune function and carcinogenesis. Taken together, both BPA and BPS are potentially carcinogenic and have deleterious effects on the fertility of laying chickens by inducing inflammation, suggesting that BPS may not be a safe replacement for BPA.

Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets.

Poultry production plays an important role in the economy and livelihoods of rural households in Kenya. As part of a surveillance program, avian influenza virus (AIV)-specific real-time RT-PCR (RRT-PCR) was used to screen 282 oropharyngeal swabs collected from chickens at six live bird markets (LBMs) and 33 backyard poultry farms in Kenya and 8 positive samples were detected. Virus was isolated in eggs from five samples, sequenced, and identified as H9N2 low pathogenic AIV (LPAIV) G1 lineage, with highest nucleotide sequence identity (98.6-99.9%) to a 2017 Ugandan H9N2 isolate. The H9N2 contained molecular markers for mammalian receptor specificity, implying their zoonotic potential. Virus pathogenesis and transmissibility was assessed by inoculating low and medium virus doses of a representative Kenyan H9N2 LPAIV isolate into experimental chickens and exposing them to naïve uninfected chickens at 2 -days post inoculation (dpi). Virus shedding was determined at 2/4/7 dpi and 2/5 days post placement (dpp), and seroconversion determined at 14 dpi/12 dpp. None of the directly-inoculated or contact birds exhibited any mortality or clinical disease signs. All directly-inoculated birds in the low dose group shed virus during the experiment, while only one contact bird shed virus at 2 dpp. Only two directly-inoculated birds that shed high virus titers seroconverted in that group. All birds in the medium dose group shed virus at 4/7 dpi and at 5 dpp, and they all seroconverted at 12/14 dpp. This is the first reported detection of H9N2 LPAIV from Kenya and it was shown to be infectious and transmissible in chickens by direct contact and represents a new disease threat to poultry and potentially to people.

The Multifaceted Zoonotic Risk of H9N2 Avian Influenza.

Poultry-adapted H9N2 avian influenza viruses (AIVs) are commonly found in many countries in Asia, the Middle East, Africa, and Europe, and although classified as low pathogenic viruses, they are an economically important disease. Besides the importance of the disease in the poultry industry, some H9N2 AIVs are also known to be zoonotic. The disease in humans appears to cause primarily a mild upper respiratory disease, and doesn't cause or only rarely causes the severe pneumonia often seen with other zoonotic AIVs like H5N1 or H7N9. Serologic studies in humans, particularly in occupationally exposed workers, show a large number of people with antibodies to H9N2, suggesting infection is commonly occurring. Of the four defined H9N2 poultry lineages, only two lineages, the G1 and the Y280 lineages, are associated with human infections. Almost all of the viruses from humans have a leucine at position 226 (H3 numbering) of the hemagglutinin associated with a higher affinity of binding with α2,6 sialic acid, the host cell receptor most commonly found on glycoproteins in the human upper respiratory tract. For unknown reasons there has also been a shift in recent years of poultry viruses in the G1 and Y280 lineages to also having leucine instead of glutamine, the amino acid found in most avian viruses, at position 226. The G1 and Y280 poultry lineages because of their known ability to infect humans, the high prevalence of the virus in poultry in endemic countries, the lack of antibody in most humans, and the shift of poultry viruses to more human-like receptor binding makes these viruses a human pandemic threat. Increased efforts for control of the virus, including through effective vaccine use in poultry, is warranted for both poultry and public health goals.

Behavioral phenotype relates to physiological differences in immunological and stress responsiveness in reactive and proactive birds.

It has now been demonstrated in many species that individuals display substantial variation in coping styles, generally separating into two major behavioral phenotypes that appear to be linked to the degree of physiological stress responsiveness. Laying hens are perfect examples of these dichotomous phenotypes; white laying hens are reactive, flighty, and exhibit large hormonal and behavioral responses to both acute and chronic stress, while brown laying hens are proactive, exploratory, and exhibit low hormonal and behavioral responses to stress. Given the linkages between stress physiology and many other body systems, we hypothesized that behavioral phenotype would correspond to additional physiological responses beyond the stress response, in this case, immunological responses. Because corticosterone is widely known to be immunosuppressive, we predicted that the reactive white hens would show more dampened immune responses than the proactive brown hens due to their exposure to higher levels of corticosterone throughout life. To assess immune function in white and brown hens, we compared febrile responses, corticosterone elevations, feed consumption, and egg production that occurred in response an injection of lipopolysaccharide (LPS) or saline, inflammatory responses to phytohemagglutinin (PHA) injection in the toe web, innate phagocytic activity in whole blood, and antibody responses to an injection of Sheep Red Blood Cells (SRBCs). Contrary to our predictions, white hens had significantly greater swelling of the toe web in response to PHA and showed a greater inhibition of feeding and reproductive output in response to LPS. These results indicated that reactive individuals are more reactive in both stress and immunological responsiveness.

Behavioral phenotype predicts physiological responses to chronic stress in proactive and reactive birds.

Animal species display significant variation in personality traits among individuals, and two main coping styles have been identified and termed "proactive" and "reactive". Further, these coping styles appear to correlate directly with the strength of the physiological stress response exhibited by those individuals. In our study system, white laying hens are reactive, flighty, and exhibit large hormonal and behavioral responses to acute stress, while brown laying hens are proactive, exploratory, and exhibit low hormonal and behavioral responses to acute stress. The objective of the current study was to determine if personality type also corresponds to differences in multiple measures of stress when birds are subjected to a chronic stressor. We tested the responses of hens to chronic stress applied by providing feed according to an unpredictable schedule for 14 days, and measured corticosterone concentrations in circulation, expression of heat shock proteins (HSPs), molecules known to protect cells in response to stress, and the ratios of heterophils:lymphocytes in blood, two immune cells known to change in quantity in circulation during chronic stress. We predicted that white hens would show greater physiological responses to the chronic stress treatment. Plasma corticosterone levels significantly increased after 7 days of treatment and returned to baseline levels on day 14, but did not differ significantly between strains. H:L ratios, on the other hand, were significantly elevated by day 7 of treatment, and increased significantly more in brown hens than white. HSP70 and HSP90 expression levels were significantly higher after stress began in white hens than brown. Our results showed that brown hens were more reactive in one response (H:L ratios) while white hens were more reactive in another (HSP expression). These different reactions to the same stressor may represent different strategies for dealing with the same stressor.