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Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the major antigen stimulating the host's protective immune response. Here we assessed the efficacy of therapeutic monoclonal antibodies (mAbs) against Omicron variant (B.1.1.529) sublineage BA.1 variants in Syrian hamsters. Of the FDA-approved therapeutic mAbs tested (that is, REGN10987/REGN10933, COV2-2196/COV2-2130 and S309), only COV2-2196/COV2-2130 efficiently inhibited BA.1 replication in the lungs of hamsters, and this effect was diminished against a BA.1.1 variant possessing the S-R346K substitution. In addition, treatment of BA.1-infected hamsters with molnupiravir (a SARS-CoV-2 RNA-dependent RNA polymerase inhibitor) or S-217622 (a SARS-CoV-2 protease inhibitor) strongly reduced virus replication in the lungs. These findings suggest that the use of therapeutic mAbs in Omicron-infected patients should be carefully considered due to mutations that affect efficacy, and demonstrate that the antiviral compounds molnupiravir and S-217622 are effective against Omicron BA.1 variants.

Previous studies have shown that the Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses circulated widely in pigs around the world and formed multiple genotypes by acquiring non-hemagglutinin and neuraminidase segments derived from other swine influenza viruses. Swine influenza control is not a priority for the pig industry in many countries, and it is worrisome that some strains may become more pathogenic and/or transmissible during their circulation in nature. Our routine surveillance indicated that the EA H1N1 viruses obtained different internal genes from different swine influenza viruses and formed various new genotypes. In this study, we found that a naturally isolated swine influenza reassortant, A/swine/Liaoning/265/2017 (LN265), a representative strain of one of the predominant genotypes in recent years, is lethal in mice and transmissible in ferrets. LN265 contains the hemagglutinin, neuraminidase, and matrix of the EA H1N1 virus; the basic polymerase 2, basic polymerase 1, acidic polymerase (PA), and nucleoprotein of the 2009 H1N1 pandemic virus; and the nonstructural protein of the North American triple-reassortment H1N2 virus. By generating and testing a series of reassortants and mutants, we found that four gradually accumulated mutations in PA are responsible for the increased pathogenicity and transmissibility of LN265. We further revealed that these mutations increase the messenger RNA transcription of viral proteins by enhancing the endonuclease cleavage activity and viral RNA–binding ability of the PA protein. Our study demonstrates that EA H1N1 swine influenza virus became pathogenic and transmissible in ferrets by acquiring key mutations in PA and provides important insights for monitoring field strains with pandemic potential.

The highly pathogenic avian influenza H5N6 viruses are widely circulating in poultry and wild birds, and have caused 38 human infections including 21 deaths; however, the key genetic determinants of the pathogenicity of these viruses have yet to be fully investigated. Here, we characterized two H5N6 avian influenza viruses – A/duck/Guangdong/S1330/2016 (GD/330) and A/environment/Fujian/S1160/2016 (FJ/160) – that have similar viral genomes but differ markedly in their lethality in mice. GD/330 is highly pathogenic with a 50% mouse lethal dose (MLD50) of 2.5 log10 50% egg infectious doses (EID50), whereas FJ/160 exhibits low pathogenicity with an MLD50 of 7.4 log10 EID50. We explored the molecular basis for the difference in virulence between these two viruses. By using reverse genetics, we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice. We found that the HA gene of FJ/160 substantially attenuated the virulence of GD/330 and that the mutation of glycine (G) to tryptophan (W) at position 225 (H3 numbering) in HA played a key role in this function. We further found that the amino acid mutation G225W in HA decreased the acid and thermal stability and increased the pH of HA activation, thereby attenuating the H5N6 virus in mice. Our study thus identifies a novel molecular determinant in the HA protein and provides a new target for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.

Surveillance of bat betacoronaviruses is crucial for understanding their spillover potential. We isolated bat sarbecoviruses from Rhinolophus cornutus bats in multiple locations in Japan. These viruses grew efficiently in cells expressing R. cornutus angiotensin converting enzyme-2, but not in cells expressing human angiotensin converting enzyme-2, suggesting a narrow host range.

Influenza A virus infection begins with the attachment of virus particles to sialic acid-containing receptors on the surface of host cells. This attachment is mediated by the viral surface glycoprotein hemagglutinin (HA). Influenza A viruses have a wide host range, meaning they are able to infect many mammal and bird species. Influenza pandemics have been caused by viruses that contain genes from avian influenza viruses. Therefore, the infection of humans with avian influenza viruses, including avian H5Nx and H7Nx viruses, poses a huge threat to public health. These avian influenza viruses can transmit directly to humans from infected poultry, but do not spread easily among people, in part, due to differences in the receptor-binding specificities of human and avian influenza viruses. Therefore, conversion from avian- to human-type receptor-binding specificity is widely believed to be necessary for the efficient transmission of avian influenza viruses among humans. Accordingly, constant monitoring of the receptor-binding specificity of avian influenza viruses is important. In this chapter, we describe the protocol for assessing the receptor-binding specificity of influenza A viruses.

Updating vaccine strains is essential to control equine influenza. We evaluated the protective efficacy of an inactivated equine influenza vaccine derived from viruses generated by reverse genetics (RG) in horses in an experimental viral challenge study. Wild-type (WT) virus (A/equine/Tipperary/1/2019) and virus generated by RG (consisting of hemagglutinin and neuraminidase genes from A/equine/Tipperary/1/2019 and six other genes from high-growth A/Puerto Rico/8/34) were inactivated by formalin for vaccine use. Twelve 1-year-old naïve horses with no antibodies against equine influenza virus were assigned to three groups (each n = 4): control, WT, and RG. They were vaccinated twice, 4 weeks apart, and were challenged with A/equine/Tipperary/1/2019 2 weeks after the second vaccination. All four horses in the control group and one horse in the WT group had pyrexia for multiple days and respiratory illness, and one horse in the RG group had pyrexia for 2 days without respiratory illness. The mean rectal temperatures and the mean concentrations of serum amyloid A in the WT and RG groups were significantly lower than those in the control group, with no significant differences between them. The WT and RG vaccines significantly reduced viral shedding relative to the control. The protective efficacy of the RG-derived inactivated vaccine against equine influenza virus is comparable to that of the vaccine derived from WT viruses in horses. The RG technique can make it easy to update equine influenza vaccine strains.

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

Several small animal models, including mice, Syrian hamsters, guinea pigs, and ferrets are used to study the pathogenicity, transmissibility, and antigenicity of seasonal and pandemic influenza viruses. Moreover, animal models are essential for vaccination and challenge studies to evaluate the immunogenicity and protective efficacy of new vaccines. However, authentic human influenza viruses do not always replicate efficiently in these animal models. Previously, we developed a high-yield A/Puerto Rico/8/34 (PR8-HY) vaccine virus backbone that conferred an increased virus yield to several seasonal influenza vaccines in eukaryotic cells and embryonated chicken eggs. Here, we show that this PR8-HY genetic backbone also increases the replication of several seasonal influenza viruses in Syrian hamsters compared to the authentic viruses. Therefore, the PR8-HY backbone is useful for animal studies to assess the biological properties of influenza viral HA and NA.

Past pandemic influenza viruses with sustained human-to-human transmissibility have emerged from animal influenza viruses. Employment of experimental models to assess the pandemic risk of emerging zoonotic influenza viruses provides critical information supporting public health efforts. Ferret transmission experiments have been utilized to predict the human-to-human transmission potential of novel influenza viruses. However, small sample sizes and a lack of standardized protocols can introduce interlaboratory variability, complicating interpretation of transmission experimental data. To assess the range of variation in ferret transmission experiments, a global exercise was conducted by 11 laboratories using two common stock H1N1 influenza viruses with different transmission characteristics in ferrets. Parameters known to affect transmission were standardized, including the inoculation route, dose, and volume, as well as a strict 1:1 donor/contact ratio for respiratory droplet transmission. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored and analyzed. The overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. Among environmental parameters that varied across laboratories, donor-to-contact airflow directionality was associated with increased transmissibility. To attain high confidence in identifying viruses with moderate to high transmissibility or low transmissibility under a smaller number of participating laboratories, our analyses support the notion that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment. IMPORTANCE Following detection of a novel virus, rapid characterization efforts (both in vitro and in vivo) are undertaken at numerous laboratories worldwide to evaluate the relative risk posed to human health. Aggregation of these data are critical, but the use of nonstandardized protocols can make interpretation of divergent results a challenge. For evaluation of virus transmissibility, a multifactorial trait which can only be evaluated in vivo, identifying intrinsic levels of variability between groups can improve the utility of these data, as well as ensure that experiments are performed with sufficient replication to ensure high confidence in compiled results. Using the ferret transmission model and two influenza A viruses, we conducted a multicenter standardization exercise to improve the interpretation of transmission data generated during risk assessment activities; this exercise serves as a model for future efforts employing both in vitro and in vivo models against possible pandemic pathogens.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced following infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases (NIAID) within the National Institutes of Health (NIH) established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants potentially impacting transmission, virulence, and resistance to convalescent and vaccine-induced immunity. The SAVE program serves as a critical data-generating component of the United States Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines, and therapeutics and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity, and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models, and pivotal findings facilitated by this collaborative approach and identify future challenges. This program serves as a template for the response against rapidly evolving pandemic pathogens by monitoring viral evolution in the human population to identify variants that could erode the effectiveness of countermeasures.

Severe acute respiratory syndrome coronavirus 2 readily transmits between domestic cats. We found that domestic cats that recover from an initial infection might be protected from reinfection. However, we found long-term persistence of inflammation and other lung lesions after infection, despite a lack of clinical symptoms and limited viral replication in the lungs.

Low pathogenic H9N2 avian influenza viruses have spread in wild birds and poultry worldwide. Recently, the number of human cases of H9N2 virus infection has increased in China and other countries, heightening pandemic concerns. In Japan, H9N2 viruses are not yet enzootic; however, avian influenza viruses, including H5N1, H7N9, H5N6, and H9N2, have been repeatedly detected in raw poultry meat carried by international flight passengers from Asian countries to Japan. Although H9N2 virus-contaminated poultry products intercepted by the animal quarantine service at the Japan border have been characterized in chickens and ducks, the biological properties of those H9N2 viruses in mammals remain unclear. Here, we characterized the biological features of two H9N2 virus isolates [A/chicken/Japan/AQ-HE28-50/2016 (Ck/HE28-50) and A/chicken/Japan/AQ-HE28-57/2016 (Ck/HE28-57)] in a mouse model. We found that these H9N2 viruses replicate well in the respiratory tract of infected mice without adaptation, and that Ck/HE28-57 caused body weight loss in the infected mice. Our results indicate that H9N2 avian influenza viruses isolated from raw chicken meat products illegally brought to Japan can potentially infect and cause disease in mammals.

Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2) in prophylactic and therapeutic settings. Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human FcγR transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in α2,6-linkage (α2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus., Here, Kuroda et al. screen a library of nearly 400 interferon-stimulated genes (ISGs) and identify several ISGs that inhibit Ebola virus entry, viral transcription/replication, or virion formation. The study provides insights into interactions between Ebola and the host cells.

The avian influenza A(H7N9) virus has caused high mortality rates in humans, especially in the elderly; however, little is known about the mechanistic basis for this. In the current study, we used nonhuman primates to evaluate the effect of aging on the pathogenicity of A(H7N9) virus. We observed that A(H7N9) virus infection of aged animals (defined as age 20–26 years) caused more severe symptoms than infection of young animals (defined as age 2–3 years). In aged animals, lung inflammation was weak and virus infection was sustained. Although cytokine and chemokine expression in the lungs of most aged animals was lower than that in the lungs of young animals, 1 aged animal showed severe symptoms and dysregulated proinflammatory cytokine and chemokine production. These results suggest that attenuated or dysregulated immune responses in aged animals are responsible for the severe symptoms observed among elderly patients infected with A(H7N9) virus.

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus–infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus–infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo., Infected lung is analyzed by two-photon imaging microscopy and can be observed for >4 h during the acute phase of inflammation and at for least 1 h in the lethal phase.

Background Here, we genetically and antigenically analyzed influenza B viruses (IBVs) isolated in Japan during the 2017‐2018 and 2018‐2019 influenza seasons. Methods A total of 68 IBVs (61 B/Yamagata/16/88‐like [B/Yamagata]‐lineage and 7 B/Victoria/2/87‐like [B/Victoria]‐lineage) were antigenically and genetically characterized by using hemagglutination inhibition (HI) assays and phylogenetic analysis, respectively. The susceptibility of IBVs to neuraminidase (NA) inhibitors was assessed by using a fluorescence‐based NA inhibition assay. Results All 61 B/Yamagata‐lineage isolates were genetically closely related to B/Phuket/3073/2013, the vaccine strain for these two seasons. Eleven B/Yamagata‐lineage isolates tested were antigenically similar to B/Phuket/3073/2013 by the HI test. Seven B/Victoria‐lineage isolates were genetically closely related to B/Texas/02/2013, the WHO‐recommended vaccine strain for the 2017‐2018 season; however, they were antigenically distinct from B/Texas/02/2013 with an eightfold or 16‐fold difference in HI titer. Of these 7 isolates, 4 possessed a two‐amino‐acid deletion at positions 162 and 163 in hemagglutinin (HA) and the other 3 had a three‐amino‐acid deletion at positions 162‐164 in HA. Importantly, the variants with the three‐amino‐acid deletion appeared to be antigenically different from the B/Colorado/06/2017 virus with the two‐amino‐acid deletion, the vaccine strain for the 2018‐2019 season with a fourfold or eightfold difference in HI titer. One B/Yamagata‐lineage isolate carrying a G407S mutation in its NA showed a marked reduction in susceptibility to zanamivir, peramivir, and laninamivir. Conclusions These results highlight the need for continued monitoring for the prevalence of the antigenic variant with the three‐amino‐acid deletion and the variant with reduced NA inhibitor susceptibility.

Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, variants with constellations of mutations in the spike gene jeopardize their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine interferon lambda (IFN-λ) has been proposed as a possible treatment based on studies in human coronavirus 2019 (COVID-19) patients. Here, we show that IFN-λ protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron) variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally delivered IFN-λ2 limits infection of historical or variant SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-λ is produced preferentially in epithelial cells and acts on radio-resistant cells to protect against SARS-CoV-2 infection. Thus, inhaled IFN-λ may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.

The emergence of the SARS-CoV-2 Delta variant (B.1.617.2) raises concerns about potential reduced sensitivity of the virus to antibody neutralization and subsequent vaccine breakthrough infections. Here, we use a live virus neutralization assay with sera from Pfizer- and Moderna-vaccinated individuals to examine neutralizing antibody titers against SARS-CoV-2 and observe a 3.9- and 2.7-fold reduction, respectively, in neutralizing antibody titers against the Delta variant compared with an early isolate bearing only a D614G substitution in its spike protein. We observe similar reduced sensitivity with sera from hamsters that were previously infected with an early isolate of SARS-CoV-2. Despite this reduction in neutralizing antibody titers against the Delta variant, hamsters previously infected (up to 15 months earlier) with an early isolate are protected from infection with the Delta variant, suggesting that the immune response to the first infection is sufficient to provide protection against subsequent infection with the Delta variant., Graphical abstract, As SARS-CoV-2 variants accumulate mutations, there is a risk of ineffective neutralizing antibodies against new variants and potential re-infection. Halfmann et al. report that, in the hamster model, previous infection with an early prototypical SARS-CoV-2 isolate prevents re-infection of the Delta variant and its transmission to naive hamsters.

Background Immunocompromised patients infected with influenza virus require prolonged treatment with neuraminidase inhibitors, because these patients are not able to eradicate the virus from the respiratory tract, leading to the emergence of drug-resistant mutant viruses. Methods In this study, we examined the efficacy of baloxavir marboxil in nude mice that were immunologically deficient. Results Daily treatment with a suboptimal dose of baloxavir marboxil increased the survival time of the virus-infected nude mice but did not clear the virus from their respiratory organs, resulting in gradual body weight loss after termination of treatment. Conclusions Despite the prolonged baloxavir marboxil treatment, few resistant mutants were detected.

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi-basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ-HE29-22/2017 (HE29-22) and A/duck/Japan/AQ-HE29-52/2017 (HE29-52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ-HE28-3/2016 (HE28-3)] in mice and ferrets. In mice, HE29-52 was more pathogenic than HE29-22 and HE28-3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, which ranges from fatal disease in some to mild or subclinical in most affected individuals. Many recovered human patients report persistent respiratory signs; however, lung disease in post-acute infection is poorly understood. Our objective was to describe histologic lung lesions and viral loads following experimental SARS-CoV-2 infection in 11 cats. Microscopic evaluation at 3, 6, 10, or 28 days postinoculation (DPI) identified mild to moderate patchy interstitial pneumonia, bronchiolar epithelial damage, and occlusive histiocytic bronchiolitis. Based on immunohistochemistry, alveolar septal thickening was due to CD204-positive macrophages, fewer B and T lymphocytes, type II pneumocytes, and capillary proliferation with a relative dearth of fibrosis. In blood vessel endothelium, there was reactive hypertrophy or vacuolar degeneration and increased MHC II expression at all time points. Unexpectedly, one cat from the 28 DPI group had severe subacute regionally extensive lymphohistiocytic pneumonia with multifocal consolidation, vasculitis, and alveolar fibrin. Reverse transcriptase-quantitative polymerase chain reaction identified SARS-CoV-2 RNA within the lung at 3 and 6 DPI, and viral RNA was below the limit of detection at 10 and 28 DPI, suggesting that pulmonary lesions persist beyond detection of viral RNA. These findings clarify our comparative understanding of disease induced by SARS-CoV-2 and suggest that cats can serve as an informative model to study post-acute pulmonary sequelae.

SUMMARYThe recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Human influenza viruses evade host immune responses by accumulating mutations around the receptor-binding region of the hemagglutinin (HA) protein, which is composed of three key elements, the 130-loop, the 190-helix, and the 220-loop. Here, we characterized two human H3N2 influenza viruses with 12- and 16-amino acid deletions around the HA receptor-binding site that were isolated after antigenic selection of mutated H3N2 viruses. Structural modeling suggested that the 12-amino acid deletion eliminated the 190-helix. The 16-amino acid deletion comprises two stretches of 11- and 5-amino acid deletions. As the result of a frameshift, “novel” amino acids (not found in wild-type HA at these positions) are encoded between the deleted regions. Interestingly, structural modeling predicted that the novel sequence forms a structure resembling the 190-helix. However, compared to wild-type HA, the 16-amino acid deletion mutant lacks two antiparallel beta-sheets that connect the 190-helix and the 220-loop in wild-type HA. Nonetheless, both HA deletion mutants replicated in mammalian cells, and the 16-amino acid deletion mutant (with a remodeled 190-helix) also replicated in Syrian hamsters, albeit at low titers. Wild-type virus bound preferentially to α2,6-linked sialic acids, whereas both mutants gained affinity for α2,3-linked sialic acids. Moreover, the 12- and 16-amino acid deletions may affect the antigenic properties of the viruses. Thus, viruses with sizeable deletions around the HA receptor-binding site are viable but may display altered sialic acid preferences, altered antigenic properties, and attenuated replicative ability in cultured cells and virulence in Syrian hamsters. IMPORTANCE The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated. In cultured cells, 12- and 16-amino acid deletion mutants were attenuated, and the 16-amino acid deletion mutant replicated in Syrian hamsters. Compared with wild-type virus, both mutants showed changes in their reactivity to some of the sera tested and changes in their binding affinity to sialic acids, which serve as influenza virus receptors. Collectively, our findings highlight the plasticity of HA.

Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19. Importance Convalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients., Endogenous ACE2 is a receptor for SARS-CoV-2 and a recombinant soluble ACE2 protein can inhibit SARS-CoV-2 infection acting as a decoy. Here the authors show that B38-CAP, an ACE2-like enzyme but not a decoy for the virus, is protective against SARS-CoV-2-induced lung injury in animal models.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

The COVID-19 pandemic continues to cause morbidity and mortality worldwide. Most approved COVID-19 vaccines generate a neutralizing antibody response that primarily targets the highly variable receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. SARS-CoV-2 "variants of concern" have acquired mutations in this domain allowing them to evade vaccine-induced humoral immunity. Recent approaches to improve the breadth of protection beyond SARS-CoV-2 have required the use of mixtures of RBD antigens from different sarbecoviruses. It may therefore be beneficial to develop a vaccine in which the protective immune response targets a more conserved region of the S protein.Here we have developed a vaccine based on the conserved S2 subunit of the S protein and optimized the adjuvant and immunization regimen in Syrian hamsters and BALB/c mice. We have characterized the efficacy of the vaccine against SARS-CoV-2 variants and other coronaviruses.Immunization with S2-based constructs elicited a broadly cross-reactive IgG antibody response that recognized the spike proteins of not only SARS-CoV-2 variants, but also SARS-CoV-1, and the four endemic human coronaviruses. Importantly, immunization reduced virus titers in respiratory tissues in vaccinated animals challenged with SARS-CoV-2 variants B.1.351 (beta), B.1.617.2 (delta), and BA.1 (omicron) as well as a pangolin coronavirus.These results suggest that S2-based constructs can elicit a broadly cross-reactive antibody response resulting in limited virus replication, thus providing a framework for designing vaccines that elicit broad protection against coronaviruses.NIH, Japan Agency for Medical Research and Development, Garry Betty/ V Foundation Chair Fund, and NSF.

The coronavirus disease 2019 (COVID-19) pandemic has raised concerns about the detrimental effects of antibodies. Antibody-dependent enhancement (ADE) of infection is one of the biggest concerns in terms of not only the antibody reaction to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) upon reinfection with the virus but also the reaction to COVID-19 vaccines. In this study, we evaluated ADE of infection by using COVID-19 convalescent-phase plasma and BHK cells expressing human Fcγ receptors (FcγRs). We found that FcγRIIA and FcγRIIIA mediated modest ADE of infection against SARS-CoV-2. Although ADE of infection was observed in monocyte-derived macrophages infected with SARS-CoV-2, including its variants, proinflammatory cytokine/chemokine expression was not upregulated in macrophages. SARS-CoV-2 infection thus produces antibodies that elicit ADE of infection, but these antibodies do not contribute to excess cytokine production by macrophages. IMPORTANCE Viruses infect cells mainly via specific receptors at the cell surface. Antibody-dependent enhancement (ADE) of infection is an alternative mechanism of infection for viruses to infect immune cells that is mediated by antibodies and IgG receptors (FcγRs). Because ADE of infection contributes to the pathogenesis of some viruses, such as dengue virus and feline coronavirus, it is important to evaluate the precise mechanism of ADE and its contribution to the pathogenesis of SARS-CoV-2. Here, using convalescent-phase plasma from COVID-19 patients, we found that two types of FcγRs, FcγRIIA and FcγRIIIA, mediate ADE of SARS-CoV-2 infection. Although ADE of infection was observed for SARS-CoV-2 and its recent variants, proinflammatory cytokine production in monocyte-derived macrophages was not upregulated. These observations suggest that SARS-CoV-2 infection produces antibodies that elicit ADE of infection, but these antibodies may not be involved in aberrant cytokine release by macrophages during SARS-CoV-2 infection.

There is an unmet need for safe and effective severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines that are stable and can be cost‐effectively produced at large scale. Here, a biopolymer particle (BP) vaccine technology that can be quickly adapted to new and emerging variants of SARS‐CoV‐2 is used. Coronavirus antigen‐coated BPs are described as vaccines against SARS‐CoV‐2. The spike protein subunit S1 or epitopes from S and M proteins (SM) plus/minus the nucleocapsid protein (N) are selected as antigens to either coat BPs during assembly inside engineered Escherichia coli or BPs are engineered to specifically ligate glycosylated spike protein (S1‐ICC) produced by using baculovirus expression in insect cell culture (ICC). BP vaccines are safe and immunogenic in mice. BP vaccines, SM‐BP‐N and S1‐ICC‐BP induced protective immunity in the hamster SARS‐CoV‐2 infection model as shown by reduction of virus titers up to viral clearance in lungs post infection. The BP platform offers the possibility for rapid design and cost‐effective large‐scale manufacture of ambient temperature stable and globally available vaccines to combat the coronavirus disease 2019 (COVID‐19) pandemic., Ambient temperature stable, scalable coronavirus disease 2019 polymer particle vaccines are developed by engineering bacterial cell factories to assemble biopolymer particles either directly coated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) antigens or to capture soluble spike protein S1 subunit produced by an insect culture. Polymer particle vaccines induce protective immunity in a hamster SARS‐CoV‐2 infection model reducing virus titers up to viral clearance in lungs post infection.

Significance COVID-19 has shown severe pathogenicity in people with underlying diseases and in elderly people. It is necessary to elucidate the pathological conditions in patients with underlying diseases and in elderly patients. The use of appropriate animal models of COVID-19 is required for the development of pharmaceutical products; however, usually healthy young animals are used as experimental animals. Cynomolgus macaques with various clinical conditions and ages were infected with severe acute respiratory syndrome coronavirus 2 and there was a difference in pathological condition between young individuals and old-aged individuals with underlying diseases; therefore, the COVID-19 cynomolgus monkey model reflecting the pathophysiology of humans would be useful for elucidating the pathophysiology and developing therapeutic and prophylactic agents., The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.

COVID-19, caused by SARS-CoV-2, has spread worldwide with dire consequences. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG promoter-driven human angiotensin-converting enzyme 2-transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. Analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were integrated in tandem into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T-cell based vaccines against respiratory viral pathogens such as influenza A virus (IAV). Chemokine receptor 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs, to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendrtitic cells (DCs) and monocyte-derived DCs internalized and processed vaccine antigen in lungs. We also found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127HI/KLRG-1LO, OX40+veCD62L+ve and mucosally imprinted CD69+veCD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs, induced by CCR2 deficiency was linked to dampened expression of T-bet, but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens including IAV and SARS-CoV-2.ImportanceWhile antibody-based immunity to influenza A virus (IAV) is type and sub-type specific, lung and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T-cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses such as IAV and SARS-CoV-2.

The effects of simultaneous or serial infections with SARS-CoV-2 and influenza virus are unknown. Here, we report that, in a hamster model, SARS-CoV-2 infection at the same time as or prior to H3N2 influenza virus infection resulted in significantly reduced influenza virus titers in the lungs and nasal turbinates of infected animals. This interference of influenza virus replication may correlate with SARS-CoV-2-induced expression of the antiviral gene MX1.

Significance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are of concern, with the P.1 variants dominating in Brazil. Brazil is now seeing a record number of deaths. Here, we report that the pathogenicity in hamsters of a P.1 variant is similar to that of nonvariant SARS-CoV-2. However, it has an expanded host range as shown by its replication in mice. Prior infection with nonvariant SARS-CoV-2 strains efficiently prevented replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. Convalescent sera from patients infected with nonvariants or sera from messenger RNA vaccinees showed comparable neutralization titers among the P.1 and previously circulating strains. These results suggest that previous SARS-CoV-2 infection and vaccines based on the original SARS-CoV-2 will provide some protection against P.1 infection., The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

The COVID-19 pandemic continues to wreak havoc as worldwide SARS-CoV-2 infection, hospitalization, and death rates climb unabated. Effective vaccines remain the most promising approach to counter SARS-CoV-2. Yet, while promising results are emerging from COVID-19 vaccine trials, the need for multiple doses and the challenges associated with the widespread distribution and administration of vaccines remain concerns. Here, we engineered the coat protein of the MS2 bacteriophage and generated nanoparticles displaying multiple copies of the SARS-CoV-2 spike (S) protein. The use of these nanoparticles as vaccines generated high neutralizing antibody titers and protected Syrian hamsters from a challenge with SARS-CoV-2 after a single immunization with no infectious virus detected in the lungs. This nanoparticle-based vaccine platform thus provides protection after a single immunization and may be broadly applicable for protecting against SARS-CoV-2 and future pathogens with pandemic potential., Chiba et al. describe the use of MS2 bacteriophage coat proteins to develop nanocarriers that display the SARS-CoV-2 spike proteins multivalently for vaccine applications. The vaccine elicited high neutralizing antibody titers and protected Syrian hamsters from virus infection after a single immunization.

Viruses are believed to be ubiquitous; however, the diversity of viruses is largely unknown because of the bias of previous research toward pathogenic viruses. Deep sequencing is a promising and unbiased approach to detect viruses from animal-derived materials. Although cranes are known to be infected by several viruses such as influenza A viruses, previous studies targeted limited species of viruses, and thus viruses that infect cranes have not been extensively studied. In this study, we collected crane fecal samples in the Izumi plain in Japan, which is an overwintering site for cranes, and performed metagenomic shotgun sequencing analyses. We detected aviadenovirus-like sequences in the fecal samples and tentatively named the discovered virus crane-associated adenovirus 1 (CrAdV-1). We determined that our sequence accounted for approximately three-fourths of the estimated CrAdV-1 genome size (33,245 bp). The GC content of CrAdV-1 genome is 34.1%, which is considerably lower than that of other aviadenoviruses. Phylogenetic analyses revealed that CrAdV-1 clusters with members of the genus Aviadenovirus, but is distantly related to the previously identified aviadenoviruses. The protein sequence divergence between the DNA polymerase of CrAdV-1 and those of other aviadenoviruses is 45.2-46.8%. Based on these results and the species demarcation for the family Adenoviridae, we propose that CrAdV-1 be classified as a new species in the genus Aviadenovirus. Results of this study contribute to a deeper understanding of the diversity and evolution of viruses and provide additional information on viruses that infect cranes, which might lead to protection of the endangered species of cranes.

Avian influenza virus (AIV) is an extraordinarily diverse pathogen that causes significant morbidity in domesticated poultry populations and threatens human life with looming pandemic potential. Controlling avian influenza in susceptible populations requires highly effective, economical and broadly reactive vaccines. Several AIV vaccines have proven insufficient despite their wide use, and better technologies are needed to improve their immunogenicity and broaden effectiveness. Previously, we developed a “mosaic” H5 subtype hemagglutinin (HA) AIV vaccine and demonstrated its broad protection against diverse highly pathogenic H5N1 and seasonal H1N1 virus strains in mouse and non-human primate models. There is a significant interest in developing effective and safe vaccines against AIV that cannot contribute to the emergence of new strains of the virus once circulating in poultry. Here, we report on the development of an H5 mosaic (H5M) vaccine antigen formulated with polyanhydride nanoparticles (PAN) that provide sustained release of encapsulated antigens. H5M vaccine constructs were immunogenic whether delivered by the modified virus Ankara (MVA) strain or encapsulated within PAN. Both humoral and cellular immune responses were generated in both specific-pathogen free (SPF) and commercial chicks. Importantly, chicks vaccinated by H5M constructs were protected in terms of viral shedding from divergent challenge with a low pathogenicity avian influenza (LPAI) strain at 8 weeks post-vaccination. In addition, protective levels of humoral immunity were generated against highly pathogenic avian influenza (HPAI) of the similar H5N1 and genetically dissimilar H5N2 viruses. Overall, the developed platform technologies (MVA vector and PAN encapsulation) were safe and provided high levels of sustained protection against AIV in chickens. Such approaches could be used to design more efficacious vaccines against other important poultry infections.

Both influenza A and B viruses cause outbreaks of seasonal influenza resulting in significant morbidity and mortality. There are two antigenically distinct lineages of influenza B virus, Yamagata lineage (YL) and Victoria lineage (VL). Since both B lineages have been co-circulating for years, more than 70% of influenza vaccines currently manufactured are quadrivalent consisting of influenza A (H1N1), influenza A (H3N2), influenza B (YL) and influenza B (VL) antigens. Although quadrivalent influenza vaccines tend to elevate immunity to both influenza B lineages, estimated overall vaccine efficacy against influenza B is still only around 42%. Thus, a more effective influenza B vaccine is needed. To meet this need, we generated BM2-deficient, single-replication (BM2SR) influenza B vaccine viruses that encode surface antigens from influenza B/Wisconsin/01/2010 (B/WI01, YL) and B/Brisbane/60/2008 (B/Bris60, VL) viruses. The BM2SR-WI01 and BM2SR-Bris60 vaccine viruses are replication-deficient in vitro and in vivo, and can only replicate in a cell line that expresses the complementing BM2 protein. Both BM2SR viruses were non-pathogenic to mice, and vaccinated animals showed elevated mucosal and serum antibody responses to both Yamagata and Victoria lineages in addition to cellular responses. Serum antibody responses included lineage-specific hemagglutinin inhibition antibody (HAI) responses as well as responses to the stem region of the hemagglutinin (HA). BM2SR vaccine viruses provided apparent sterilizing immunity to mice against intra- and inter-lineage drifted B virus challenge. The data presented here support the feasibility of BM2SR as a platform for next-generation trivalent influenza vaccine development.

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine.

Here, we developed hCK, a Madin-Darby canine kidney (MDCK) cell line that expresses high levels of human influenza virus receptors and low levels of avian virus receptors. hCK cells supported human A/H3N2 influenza virus isolation and growth much more effectively than conventional MDCK or human virus receptor-overexpressing (AX4) cells. A/H3N2 viruses propagated in hCK cells also maintained higher genetic stability than those propagated in MDCK and AX4 cells.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune-evasion properties have arisen, which could jeopardize recently deployed vaccine- and antibody-based countermeasures.Here, we evaluated in mice and hamsters the efficacy of a pre-clinical version of the Moderna mRNA vaccine (mRNA-1273) and the JohnsonJohnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N.Immunization of 129S2 and K18-human ACE2 transgenic mice with the mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with the B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge.Thus, immunity induced by the mRNA-1273 or Ad26.COV2.S vaccine can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.This study was supported by the NIH (R01 AI157155 and U01 AI151810), NIAID Centers of Excellence for Influenza Research and Response [CEIRR] contracts 75N93021C00014 and 75N93021C00016, and the Collaborative Influenza Vaccine Innovation Centers [CIVIC] contract 75N93019C00051. It was also supported, in part, by the National Institutes of Allergy and Infectious Diseases Center for Research on Influenza Pathogenesis (HHSN272201400008C) and the Japan Program for Infectious Diseases Research and Infrastructure (JP21wm0125002) from the Japan Agency for Medical Research and Development (AMED).

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies (MAbs) targeted to the hemagglutinin (HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.

The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants., Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.

Background Keeping vaccine strains up to date is the key to controlling equine influenza (EI). Viruses generated by reverse genetics (RG) are likely to be effective for quickly updating a vaccine strain. Objectives To evaluate the growth properties of an RG virus in embryonated chicken eggs, and to evaluate antibody responses to a formalin-inactivated vaccine derived from the RG virus in Thoroughbred horses. Study design In vitro and in vivo experiments. Methods Wild-type (WT) viruses (A/equine/Ibaraki/1/2007) or RG viruses (consisting of haemagglutinin [HA] and neuraminidase genes derived from A/equine/Ibaraki/1/2007 and the six other genes derived from high-growth A/Puerto Rico/8/34) were inoculated into embryonated chicken eggs, and the allantoic fluids were harvested at every 24 hours after inoculation. WT and RG viruses were inactivated by formalin for vaccine use. Ten unvaccinated yearlings (five each for WT or RG vaccine) received the first two doses of a primary vaccination course 4 weeks apart followed by their third dose 12 weeks later. Twenty vaccinated adult horses (10 each for WT or RG vaccine) received a single dose of a booster vaccination. Results The RG virus had high growth properties in embryonated chicken eggs. Unvaccinated yearlings responded poorly to the first vaccination, especially those that received the RG vaccine, but mounted better responses to the second and the third vaccinations, and maintained relatively high haemagglutination inhibition (HI) titres up to 28 weeks after the first vaccination. Vaccinated adult horses did not respond remarkably to the booster vaccination, but no horses showed titres below their pre-booster values even at 12 weeks after vaccination. The RG virus elicited immunogenicity in horses adequate for vaccine use. Main limitations No virus challenge study was performed. Conclusions The RG viruses are useful as an EI vaccine strain, and quick updates of an EI vaccine strain can be achieved by using RG techniques.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems., Author summary Through ongoing human adaptation, spill-back events from other animal intermediates, or with the distribution of vaccines and therapeutics, the landscape of SARS-CoV-2 genetic variation is certain to change. The evolutionary mechanisms by which SARS-CoV-2 will continue to adapt to mammalian hosts depend on genetic variation generated within and between hosts. Here, using domestic cats as a model, we show that within-host SARS-CoV-2 genetic variation is predominantly influenced by genetic drift and purifying selection. Transmission of SARS-CoV-2 between hosts is defined by a narrow transmission bottleneck, involving 2–5 viruses. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which arises rapidly and is transmitted in cats. Spike H655Y has been previously shown to confer escape from human monoclonal antibodies and is currently found in over 1,000 human sequences. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge, underscoring the importance of continued genomic surveillance in humans and non-human mammalian hosts.