Host Subcellular Organelles: Targets of Viral Manipulation.

Viruses have evolved sophisticated mechanisms to manipulate host cell processes and utilize intracellular organelles to facilitate their replication. These complex interactions between viruses and cellular organelles allow them to hijack the cellular machinery and impair homeostasis. Moreover, viral infection alters the cell membrane's structure and composition and induces vesicle formation to facilitate intracellular trafficking of viral components. However, the research focus has predominantly been on the immune response elicited by viruses, often overlooking the significant alterations that viruses induce in cellular organelles. Gaining a deeper understanding of these virus-induced cellular changes is crucial for elucidating the full life cycle of viruses and developing potent antiviral therapies. Exploring virus-induced cellular changes could substantially improve our understanding of viral infection mechanisms.

Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks.

For many viruses, narrow bottlenecks acting during transmission sharply reduce genetic diversity in a recipient host relative to the donor. Since genetic diversity represents adaptive potential, such losses of diversity are though to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics during transmission is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a novel barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process diversity is lost for influenza A viruses. In inoculated guinea pigs, we show that a high level of viral genetic diversity is maintained across time. Continuity in the barcodes detected furthermore indicates that stochastic effects are not pronounced within inoculated hosts. Importantly, in both aerosol-exposed and direct contact-exposed animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a predominant role in this process. This finding suggests that immune selection may have greater opportunity to operate during influenza A transmission than previously recognized.

Evolution and international transmission of H3N2 canine influenza A viruses from Korea during 2014-2017.

Avian-origin H3N2 canine influenza A viruses (CIVs) have become enzootic in China and Korea and have sporadically transmitted to North America, causing multiple epidemics. We isolated six CIVs in Korea from CIV-infected patients during 2014-2017 and conducted whole genome sequencing and phylogenetic analyses. Results revealed that CIVs have circulated and evolved in Korea since the early 2000s and then diversified into a new clade, probably contributing to multiple epidemics in China, the USA, and Canada. Our findings bridge an evolutionary gap for understanding the global transmission of CIVs, emphasizing the significance of continuous monitoring of CIVs.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Beneficial effects of cellular coinfection resolve inefficiency in influenza A virus transcription.

For diverse viruses, cellular infection with single vs. multiple virions can yield distinct biological outcomes. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells. Here, we sought to uncover the viral processes underlying this phenotype. We found that the need for multiple infection maps to amino acid 26K of the viral PA protein. PA 26K suppresses endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. In the context of the higher functioning PA 26E, inhibition of PA using baloxavir acid augments reliance on multiple infection. Together, these data suggest a model in which sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral ribonucleoprotein templates to the cell. More broadly, the finding that deficiency in a core viral function is ameliorated through multiple infection suggests that the fitness effects of many viral mutations are likely to be modulated by multiplicity of infection, such that the shape of fitness landscapes varies with viral densities.

Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission.

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing and high inoculation dose. However, contrary to expectation, trends observed with sub-optimal exposure timing and lower inoculation dose suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety.

For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3'-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.

Animal models for SARS-CoV-2.

Since its first detection in December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide, resulting in over 79.2 million documented cases in one year. Lack of pre-existing immunity against this newly emerging virus has pushed the urgent development of anti-viral therapeutics and vaccines to reduce the spread of the virus and alleviate disease. Appropriate animal models recapitulating the pathogenesis of and host responses to SARS-CoV-2 infection in humans have and will continue to accelerate this development process. Several animal models including mice, hamsters, ferrets, and non-human primates have been evaluated and actively applied in preclinical studies. However, since each animal model has unique features, it is necessary to weigh the strengths and weaknesses of each according to the goals of the study. Here, we summarize the key features, strengths and weaknesses of animal models for SARS-CoV-2, focusing on their application in anti-viral therapeutic and vaccine development.

Collective interactions augment influenza A virus replication in a host-dependent manner.

Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required in a given cell. Here, we show that the reliance of IAV on multiple infection can form an important species barrier. Namely, we find that avian H9N2 viruses representative of those circulating widely at the poultry-human interface exhibit acute dependence on collective interactions in mammalian systems. This need for multiple infection is greatly reduced in the natural host. Quantification of incomplete viral genomes showed that their complementation accounts for the moderate reliance on multiple infection seen in avian cells but not the added reliance seen in mammalian cells. An additional form of virus-virus interaction is needed in mammals. We find that the PA gene segment is a major driver of this phenotype and that both viral replication and transcription are affected. These data indicate that multiple distinct mechanisms underlie the reliance of IAV on multiple infection and underscore the importance of virus-virus interactions in IAV infection, evolution and emergence.

Rank orders of mammalian pathogenicity-related PB2 mutations of avian influenza A viruses.

The PB2 gene is one of the key determinants for the mammalian adaptation of avian influenza A viruses (IAVs). Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different genetic backgrounds of avian IAVs, the relative effects of single or multiple mutations on viral fitness could not be directly compared. Furthermore, their mutational steps during mammalian adaptation had been unclear. In this study, we collectively compared the effects of individual and combined MPMs on viral fitness and determined their rank orders using a prototypic PB2 gene. Early acquired mutations may determine the function and potency of subsequent mutations and be important for recruiting multiple, competent combinations of MPMs. Higher mammalian pathogenicity was acquired with the greater accumulation of MPMs. Thus, the rank orders and the prototypic PB2 gene may be useful for predicting the present and future risks of PB2 genes of avian and mammalian IAVs.

Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin.

Clade 2.3.4.4c H5N6 avian influenza A viruses (AIVs) may have originally adapted to infect chickens and have caused highly pathogenic avian influenza (HPAI) in poultry and human fatalities. Although A/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant clade 2.3.4.4c H5N6 vaccine strains have been effective in embryonated chicken eggs-based vaccine production system, they need to be improved in terms of immunogenicity and potential mammalian pathogenicity. We replaced the PB2 gene alone or the PB2 (polymerase basic protein 2), NP (nucleoprotein), M (matrix protein) and NS (non-structural protein) genes together in the PR8 strain with corresponding genes from AIVs with low pathogenicity to remove mammalian pathogenicity and to match CD8+ T cell epitopes with contemporary HPAI viruses, respectively, without loss of viral fitness. Additionally, we tested the effect of the H103Y mutation of hemagglutinin (HA) on antigen productivity, mammalian pathogenicity and heat/acid stability. The replacement of PB2 genes and the H103Y mutation reduced the mammalian pathogenicity but increased the antigen productivity of the recombinant vaccine strains. The H103Y mutation increased heat stability but unexpectedly decreased acid stability, probably resulting in increased activation pH for HA. Interestingly, vaccination with inactivated recombinant virus with replaced NP, M and NS genes halted challenge virus shedding earlier than the recombinant vaccine without internal genes replacement. In conclusion, we successfully generated recombinant clade 2.3.4.4c H5N6 vaccine strains that were less pathogenic to mammals and more productive and heat stable than conventional PR8-derived recombinant strains by optimization of internal genes and the H103Y mutation of HA.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity.

Abstract: Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

Bioengineering a highly productive vaccine strain in embryonated chicken eggs and mammals from a non-pathogenic clade 2·3·4·4 H5N8 strain.

The clade 2·3·4·4 H5Nx is a highly pathogenic avian influenza (HPAI) virus, which first appeared in China and has spread worldwide since then, including Korea. It is divided into subclades a - d, but the PR8-derived recombinant clade 2·3·4·4 a viruses replicate inefficiently in embryonated chicken eggs (ECEs). High virus titer in ECEs and no mammalian pathogenicity are the most important prerequisites of efficacious and safer vaccine strains against HPAI. In this study, we have synthesized hemagglutinin (HA) and neuraminidase (NA) genes based on the consensus amino acid sequences of the clade 2·3·4·4a and b H5N8 HPAIVs, using the GISAID database. We generated PR8-derived H5N8 recombinant viruses with single point mutations in HA and NA, which are related to efficient replication in ECEs. The H103Y mutation in HA increased mammalian pathogenicity as well as virus titer in ECEs, by 10-fold. We also successfully eradicated mammalian pathogenicity in H103Y-bearing H5N8 recombinant virus by exchanging PB2 genes of PR8 and 01310 (Korean H9N2 vaccine strain). The final optimized H5N8 vaccine strain completely protected against a heterologous clade 2·3·4·4c H5N6 HPAIV in chickens, and induced hemagglutination inhibition (HI) antibody in ducks. However, the antibody titer of ducks showed age-dependent results. Thus, H103Y and 01310PB2 gene have been successfully applied to generate a highly productive, safe, and efficacious clade 2·3·4·4 H5N8 vaccine strain in ECEs.

Molecular evolution of Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum in the field.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.

An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex.

For reported primer sets used to detect influenza A viruses (IAVs), we verified the nucleotide identities with 9,103 complete sequences of matrix (M) genes. At best, only 93.2% and 85.3% of the sequences had a 100% match with reported forward and reverse primers, respectively. Therefore, we designed new degenerate forward and reverse primers with 100% identity to 94.4% and 96.2% of compared genes, respectively, and the primer set was used with SYBR-based reverse-transcription real-time PCR (SYBR-RT-rtPCR) for lower detection limits. The sensitivity of SYBR-RT-rtPCR with the new primers was 10-fold higher than that with a conventional method in ~2.37% of all M genes in the database used in our study. We successfully increased the sensitivity of SYBR-RT-rtPCR by concentrating the viral ribonucleoprotein (RNP) using immunomagnetic beads and Triton X-100. The improved generic primer set and RNP concentration method may be useful for sensitive detection of IAVs.

Generation of highly productive and mammalian nonpathogenic recombinant H9N2 avian influenza viruses by optimization of 3'end promoter and NS genome.

We developed A/PR/8/34 (PR8) virus-based reverse genetics system in which six internal genes of PR8 and attenuated hemagglutinin and intact neuraminidase genes of field avian influenza viruses (AIVs) have been used for the generation of highly productive recombinant vaccine strains. The 6 + 2 recombinant vaccine strains can induce protective humoral immunity against intended field AIVs; however, the epitopes of B and T cells encoded by internal genes may be important for heterosubtypic protection. Therefore, it is advantageous to use homologous internal genes of field AIVs for recombinant vaccine strains. However, the rescue of recombinant viruses having whole internal genes of field AIVs by the PR8-based reverse genetics system was unsuccessful in some cases. Although partial replacement of an internal gene has been successful for generation of highly productive and mammalian nonpathogenic recombinant viruses, complete replacement of internal genes may be more favorable. In this study, we successfully generated complete recombinant H9N2 AIVs possessing 8 genomes of H9N2 AIVs by optimal combinations of 3' end promoter sequences of polymerase genomes, and a NS genome. All the generated recombinant viruses showed highly productive and mammalian nonpathogenic traits but some of them showed much higher virus titers in embryonated chicken eggs. Additionally, we found the same mutations of NS1 gene determined pathogenicity of AIVs in chicken embryos as well as mammals. Thus, the 3' end promoter optimization, and highly productive and mammalian nonpathogenic internal genes may be useful to develop vaccines against AIVs.

Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII).

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 °C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

Acquisition of Innate Inhibitor Resistance and Mammalian Pathogenicity During Egg Adaptation by the H9N2 Avian Influenza Virus.

An H9N2 avian influenza A virus (AIV), A/chicken/Korea/01310/2001 (01310-CE20), was established after 20 passages of influenza A/chicken/Korea/01310/2001 (01310-CE2) virus through embryonated chicken eggs (ECEs). As a result of this process, the virus developed highly replicative and pathogenic traits within the ECEs through adaptive mutations in hemagglutinin (HA: T133N, V216G, and E439D) and neuraminidase (NA: 18-amino acid deletion and E54D). Here, we also established that 01310-CE20 acquired resistance to innate inhibitors present in the egg white during these passages. To investigate the role of egg-adapted mutations in resistance to innate inhibitors, we generated four PR8-derived recombinant viruses using various gene combinations of HA and NA from 01310-CE2 and 01310-CE20 (rH2N2, rH2N20, rH20N2, and rH20N20). As expected, rH20N20 showed significantly higher replication efficiency in MDCK cells and mouse lungs, and demonstrated greater pathogenicity in mice. In addition, rH20N20 showed higher resistance to innate inhibitors than the other viruses. By using a loss-of-function mutant and receptor-binding assay, we demonstrated that a T133N site directed mutation created an additional N-glycosite at position 133 in rH20N20. Further, this mutation played a crucial role in viral replication and resistance to innate inhibitors by modulating the binding affinities to avian-like and mammalian-like receptors on the host cells and inhibitors. Thus, egg-adapted HA and NA may exacerbate the mammalian pathogenicity of AIVs by defying host innate inhibitors as well as by increasing replication efficiency in mammalian cells.