Wild-type Virus Research Articles

Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging.

A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.

Efficacy of favipiravir against influenza virus resistant to both baloxavir and neuraminidase inhibitors.

Widespread resistance of influenza viruses to neuraminidase (NA) inhibitor or polymerase inhibitor, baloxavir, is a major public health concern. The amino acid mutations R152K in NA and I38T in polymerase acidic (PA) are responsible for resistance to NA inhibitors and baloxavir, respectively. We generated recombinant A(H1N1)pdm09 viruses possessing NA-R152K, PA-I38T or both mutations by using a plasmid-based reverse genetics system, characterized their virological properties in vitro and in vivo, and examined whether oseltamivir, baloxavir and favipiravir are effective against these mutant viruses. The three mutant viruses showed similar or superior growth kinetics and virulence to those of wild-type virus. Although oseltamivir and baloxavir blocked the replication of the wild-type virus in vitro, oseltamivir and baloxavir failed to suppress the replication of the NA-R152K and PA-I38T viruses in vitro, respectively. Mutant virus possessing both mutations grew in the presence of oseltamivir or baloxavir in vitro. Baloxavir treatment protected mice from lethal infection with wild-type or NA-R152K virus, but failed to protect mice from lethal infection with PA-I38T or PA-I38T/NA-R152K virus. Favipiravir treatment protected mice from lethal infection with all viruses tested, whereas oseltamivir treatment did not protect at all. Our findings indicate that favipiravir should be used to treat patients with suspected baloxavir-resistant virus infection.

The Highly Conserved Stem-Loop II Motif Is Dispensable for SARS-CoV-2.

The stem-loop II motif (s2m) is an RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over 25 years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild-type and s2m deletion viruses using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) and dimethyl sulfate mutational profiling and sequencing (DMS-MaPseq). These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'-UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. IMPORTANCE RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contain functional structures to support virus replication, translation, and evasion of the host antiviral immune response. The 3' untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is an RNA structural element that is found in many RNA viruses. This motif was discovered over 25 years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that s2m is dispensable for SARS-CoV-2.

Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP.

Human adenovirus 55 (HAdV-55) has recently caused outbreaks of acute respiratory disease (ARD), posing a significant public threat to civilians and military trainees. Efforts to develop antiviral inhibitors and quantify neutralizing antibodies require an experimental system to rapidly monitor viral infections, which can be achieved through the use of a plasmid that can produce an infectious virus. Here, we used a bacteria-mediated recombination approach to construct a full-length infectious cDNA clone, pAd55-FL, containing the whole genome of HadV-55. Then, the green fluorescent protein expression cassette was assembled into pAd55-FL to replace the E3 region to obtain a recombinant plasmid of pAd55-dE3-EGFP. The rescued recombinant virus rAdv55-dE3-EGFP is genetically stable and replicates similarly to the wild-type virus in cell culture. The virus rAdv55-dE3-EGFP can be used to quantify neutralizing antibody activity in sera samples, producing results in concordance with the cytopathic effect (CPE)-based microneutralization assay. Using an rAdv55-dE3-EGFP infection of A549 cells, we showed that the assay could be used for antiviral screening. Our findings suggest that the rAdv55-dE3-EGFP-based high-throughput assay provides a reliable tool for rapid neutralization testing and antiviral screening for HAdV-55.

A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8+ T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma

ObjectiveTo evaluate targeted killing of hepatocellular carcinoma (HCC) cells by a recombinant oncolytic influenza virus expressing a PD-L1 antibody (rgFlu/PD-L1) and to develop a novel immunotherapy for HCC. MethodsUsing influenza virus reverse genetics, a recombinant oncolytic virus was generated in the background of the A/Puerto Rico/8/34 (PR8) virus, then identified via screening and passage in specific pathogen-free chicken embryos. Hepatocellular carcinoma cell killing by rgFlu/PD-L1 was confirmed in vitro and in vivo. Transcriptome analyses were used to explore PD-L1 expression and function. Western blotting revealed that PD-L1 activated the cGas-STING pathway. ResultsrgFlu/PD-L1 expressed the PD-L1 heavy and light chain in PB1 and PA, respectively; PR8 served as the backbone. The hemagglutinin titer of rgFlu/PD-L1 was 29, and the virus titer was 9–10 logTCID50/mL. Electron microscopy revealed that the rgFlu/PD-L1 morphology and size were consistent with wild-type influenza virus. The MTS assay showed that rgFlu/PD-L1 induced significant killing of HCC cells but not normal cells. rgFlu/PD-L1 inhibited PD-L1 expression and induced apoptosis in HepG2 cells. Notably, rgFlu/PD-L1 controlled the viability and function of CD8+ T cells by activating the cGas-STING pathway. ConclusionrgFlu/PD-L1 activated the cGas-STING pathway in CD8+ T cells, causing them to kill HCC cells. This approach represents a novel immunotherapy for liver cancer.

Construction and Characterization of Severe Fever with Thrombocytopenia Syndrome Virus with a Fluorescent Reporter for Antiviral Drug Screening.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a novel bunyavirus (SFTSV) is an emerging infectious disease with up to 30% case fatality. Currently, there are no specific antiviral drugs or vaccines for SFTS. Here, we constructed a reporter SFTSV in which the virulent factor nonstructural protein (NSs) was replaced by eGFP for drug screening. First, we developed a reverse genetics system based on the SFTSV HBMC5 strain. Then, the reporter virus SFTSV-delNSs-eGFP was constructed, rescued, and characterized in vitro. SFTSV-delNSs-eGFP showed similar growth kinetics with the wild-type virus in Vero cells. We further detected the antiviral efficacy of favipiravir and chloroquine against wild-type and recombinant SFTSV by the quantification of viral RNA, and compared the results with that of fluorescent assay using high-content screening. The results showed that SFTSV-delNSs-eGFP could be used as a reporter virus for antiviral drug screening in vitro. In addition, we analyzed the pathogenesis of SFTSV-delNSs-eGFP in interferon receptor-deficient (IFNAR-/-) C57BL/6J mice and found that unlike the fatal infection of the wild-type virus, no obvious pathological change or viral replication were observed in SFTSV-delNSs-eGFP-infected mice. Taken together, the green fluorescence and attenuated pathogenicity make SFTSV-delNSs-eGFP a potent tool for the future high-throughput screening of antiviral drugs.

Expanding Opportunities in Viral Oncolysis.

Expanding Opportunities in Viral Oncolysis.

A disynaptic basal ganglia connection to the inferior olive: potential for basal ganglia influence on cerebellar learning.

Recent studies have shown that the cerebellum and the basal ganglia are interconnected at subcortical levels. However, a subcortical basal ganglia connection to the inferior olive (IO), being the source of the olivocerebellar climbing fiber system, is not known. We have used classical tracing with CTb, retrograde transneuronal infection with wildtype rabies virus, conditional tracing with genetically modified rabies virus, and examination of material made available by the Allen Brain Institute, to study potential basal ganglia connections to the inferior olive in rats and mice. We show in both species that parvalbumin-positive, and therefore GABAergic, neurons in the entopeduncular nucleus, representing the rodent equivalent of the internal part of the globus pallidus, innervate a group of cells that surrounds the fasciculus retroflexus and that are collectively known as the area parafascicularis prerubralis. As these neurons supply a direct excitatory input to large parts of the inferior olivary complex, we propose that the entopeduncular nucleus, as a main output station of the basal ganglia, provides an inhibitory influence on olivary excitability. As such, this connection may influence olivary involvement in cerebellar learning and/or could be involved in transmission of reward properties that have recently been established for olivocerebellar signaling.

CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Identification of a new polymorphism on the wild-type canine distemper virus genome: could this contribute to vaccine failures?

The canine distemper virus (CDV) is responsible for a multisystem infectious disease with high prevalence in dogs and wild carnivores and has vaccination as the main control measure. However, recent studies show an increase in cases including vaccinated dogs in different parts of the world. There are several reasons for vaccine failures, including differences between vaccine strains and wild-type strains. In this study, a phylogenetic analysis of CDV strains from samples of naturally infected, vaccinated, and symptomatic dogs in Goiânia, Goiás, Brazil was performed with partial sequencing of the hemagglutinin (H) gene of CDV. Different sites of amino acid substitutions were found, and one strain had the Y549H mutation, typically present in samples from wild animals. Substitutions in epitopes (residues 367, 376, 379, 381, 386, and 388) that may interfere with the vaccine's ability to provide adequate protection against infection for CDV were observed. The identified strains were grouped in the South America 1/Europe lineage, with a significant difference from other lineages and vaccine strains. Twelve subgenotypes were characterized, considering a nucleotide identity of at least 98% among the strains. These findings highlight the relevance of canine distemper infection and support the need better monitoring of the circulating strains that contribute to elucidate if there is a need for vaccine update.

Mechanistic insight into immune amnesia and immunity induced by wild-type measles virus compared to live-attenuated measles vaccine

Abstract Despite the availability of a safe and effective vaccine, measles still causes significant mortality and morbidity among children in developing countries. As a disease that induces immune amnesia with increased susceptibility to other infections but also life-long immunity to measles, little is known about the immunogenetics of B cells that elicit measles-specific antibodies (Abs) in natural infection vs vaccination. We therefore conducted single-cell RNA sequencing (scRNA-seq) of longitudinal peripheral blood mononuclear cells (PBMC) and lymph node (LN) samples from wild-type (WT) measles virus (MeV) and live-attenuated measles vaccine (LAMV)-infected rhesus macaques (RMs) and characterized their gene expression, B cell repertoires and clonotype changes via bioinformatic pipelines. Preliminary analysis of two RMs across four timepoints (one WT MeV- and one LAMV-infected RM) revealed similarities and differences in immune cell populations and B cell repertoires, providing important mechanistic insights into B cell clonal development in natural infection as compared to vaccination. Supported by grants from NIH R01 AI153140 and the Jeffrey Richardson Fellowship

Intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate confers potent mucosal and systemic immunity and protection

Abstract Development of effective SARS-CoV-2 vaccines to induce potent and long-lasting immunity and to provide cross-reactive protection against emerging variants remains a high priority. We recently reported a live-attenuated SARS-CoV-2 vaccine candidate with re-engineered viral transcription regulator sequences and deleted open-reading-frames (ORF) 3, 6, 7, and 8 (Δ3678) is highly attenuated in mice and hamsters and vaccination with Δ3678 SARS-CoV-2 protect hamsters from wild-type virus challenge and transmission. Here we report that a single dose intranasal vaccination with the Δ3678 SARS-CoV-2 live attenuated vaccine candidate induced strong SARS-CoV-2-specific systemic and mucosal humoral and T cell-mediated immune responses at one-month post-vaccination and day 4 post wild-type virus challenge, which are at similar levels to those infected with wild-type virus. High titer of SARS-CoV-2-specific IgG and IgA were detected in sera and BAL of vaccinated mice respectively. Furthermore, mice vaccinated with the Δ3678 vaccine showed markedly diminished viral loads and tissue inflammation in the lung following wild-type SARS-CoV-2 challenge. In summary, our results suggest that the Δ3678 SARS-CoV-2 live attenuated vaccine protects mice from wild-type SARS-CoV-2 infection by induction of protective mucosal and systemic cell-mediated and humoral responses.

Multiplex assays to determine correlation of binding and neutralizing antibodies following SARS-CoV-2 infection and vaccination

Abstract The COVID-19 pandemic has led to the rapid development of many different therapeutics and vaccines to reduce the burden of the disease. Most vaccines target the highly immunogenic spike (S) protein for generating functional immune responses to block the entry of the virus into host cells. However, the mutations in the S-protein have led to the emergence of many variants that escape the immune response. We developed two multiplex assays using Luminex® xMAP® technology to assess antibody profiles after infection and vaccination; 1) to quantify the binding antibodies against SARS-CoV2 receptor binding protein (RBD), nucleocapsid protein (NP), S1-protein (S1), and Trimer having S1, S2, and extracellular domain protein; 2) a multiplex surrogate virus neutralization antibody assay targeting CoV2 wild type and various mutant viruses. The quantitative binding assay uses the WHO standard for determining the binding antibody unit (BAU)/mL. Surrogate virus neutralization assay measures the inhibition of ACE2 protein binding to wild-type and S protein variants coupled to beads in the presence of immune or convalescent sera. We determined the negative threshold using 81 pre-pandemic serum samples collected during 2017–18. We used 116 samples collected from 45 vaccinated and 15 infected subjects for antibody quantification. Binding antibodies to RBD, S1, and trimer were higher in vaccinated individuals with minimal reactivity to NP, whereas the NP antibody response was higher in the infected individuals. We observed a direct correlation between spike protein binding and neutralizing antibodies using the two assays. We saw the most elevated neutralization antibodies against variants from individuals with breakthrough infections.

Mechanism(s) of long-term humoral immunity and immune suppression induced by wildtype Measles virus in rhesus macaques

Abstract Measles virus (MeV) causes a highly contagious systemic viral infection that remains an important cause of morbidity and mortality in children worldwide. While recovery from measles infection induces life-long protective immunity, it also causes immune amnesia which can lead to higher susceptibility to other infections. To better understand the long-term immune responses induced by the MeV vaccine versus wildtype (WT) MeV, first, we studied rhesus macaques for six months after WT MeV or Measles-Mumps-Rubella (MMR) inoculation. We quantified total antibody secreting cells (ASC) and MeV-specific ASCs in peripheral blood mononuclear cells (PBMCs) using well-characterized in house methods for ELISpot; and assessed MeV-specific IgG, avidity, and plaque reduction neutralization titers. Second, we assessed whether the loss in immune memory post WT MeV infection is linked to eviction of B cells from the bone marrow (BM) into circulation. BM and PBMCs from live attenuated MeV (LAMV) vaccinated monkeys were cultured and supernatant were assessed for isotype and antigen specificity by ELISA. PBMC cultures produced detectable amount of MeV-specific antibody and antibody recognition profiles will be compared via Virscan, a phage-display technology for epitope mapping. A novel panel of non-human primate (NHP) pathogen peptides representing Herpesviruses, Retroviruses, Adenoviruses, Pneumoviruses, and Enteroviruses were developed and expressed using the system with plans to use them to characterize changes in antibody production. We hypothesis that the robust immune response to WT MeV is accompanied by eviction of long-lived plasma cells from BM niches resulting in a loss of Ab diversity that does not occur after infection with LAMV. Supported by grants from NIH (5R01AI153140-03)

Development of an Inactivated Camelpox Vaccine from Attenuated Camelpox Virus Strain: Safety and Protection in Camels.

This article describes the preparation of an inactivated vaccine from an attenuated strain of camelpox. The attenuated camelpox virus (CMLV) was grown in lamb kidney cells and in Vero cells. CMLV was accumulated to a significantly higher (p ≤ 0.05) titer in lamb kidney cells (7.75 ± 0.08 log TCID50/mL) than in Vero cells (4.00 ± 0.14 log TCID50/mL). During virus inactivation, a concentration of 0.05% beta-propiolactone (BPL) completely inactivated the virus in 6 h at a temperature of 22 ± 1 °C, while a concentration of 0.2% formaldehyde inactivated the virus in 8 h. However, a viral antigen inactivated by BPL was used for vaccine preparation. The inactivated viral antigen was adsorbed with aluminum hydroxide gel, and as a result, an inactivated candidate vaccine was prepared. While the safety of the candidate vaccine was tested in camels and white mice, the protective efficacy of the vaccine was tested only in camels. In the safety evaluation of the inactivated vaccine, the vaccine was not observed to cause any adverse effects in mice and camels. During the immunogenicity study in camels, antibody formation started (0.2 ± 0.16 log2) at Day 21 post-vaccination (PV), and the antibody titer peaked (1.33 ± 0.21 log2) at Day 60 PV and decreased at Day 90 PV (0.50 ± 0.22 log2). Furthermore, no antibodies were detected in vaccinated camels from Days 180 to 365 PV. Camels that received vaccination and were subsequently exposed to wild-type virus evinced a healthy state despite lacking antibodies. In contrast, unvaccinated camels exhibited susceptibility to camelpox upon challenge.

Binding and avidity signatures of polyclonal sera from individuals with different exposure histories to SARS-CoV-2 infection, vaccination, and Omicron breakthrough infections.

The number of exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to vaccination antigens impact the magnitude and avidity of the polyclonal response. We studied binding and avidity of different antibody isotypes to the spike, the receptor binding domain (RBD), and the nucleoprotein (NP) of wild type (WT) and BA.1 SARS-CoV-2 in convalescent, mRNA vaccinated, mRNA boosted, hybrid immune individuals, and in individuals with breakthrough cases during the peak of the BA.1 wave. We found an increase in spike binding antibodies and antibody avidity with increasing number of exposures to infection and/or vaccination. Nucleoprotein antibodies were detectible in convalescent individuals and a proportion of breakthrough cases, but displayed low avidity. Omicron breakthrough infections elicited high levels of cross-reactive antibodies between WT and BA.1 antigens in vaccinated individuals without prior infection directed against the spike and receptor binding domain (RBDs). The magnitude of the antibody response and avidity correlated with neutralizing activity against WT virus. The magnitude and quality of the antibody response increased with the number of antigen exposures, including breakthrough infections. However, cross-reactivity of the antibody response after BA.1 breakthroughs, was impacted by the number of prior antigenic exposures.

Vaccination and Omicron BA.1/BA.2 Convalescence Enhance Systemic but Not Mucosal Immunity against BA.4/5.

Rising breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4/5 led to the performance of various studies investigating systemic immunity and neutralizing antibodies in sera, but mucosal immunity remains understudied. In this cohort study, the humoral immune responses, including immunoglobulin levels and the presence of virus-neutralizing antibodies, of 92 vaccinated and/or BA.1/BA.2 convalescent individuals were investigated. Cohorts received two doses of ChAdOx1, BNT162b2, or mRNA-1273 and subsequent booster vaccination with either BNT162b2 or mRNA-1273, following BA.1/BA.2 infection. In addition, vaccinated and nonconvalescent or unvaccinated and BA.1 convalescent individuals were studied. Serum and saliva samples were used to determine SARS-CoV-2 spike-specific IgG and IgA titers and neutralizing activity against replication-competent SARS-CoV-2 wild-type virus and the Omicron BA.4/5 variant. Vaccinated/convalescent cohorts demonstrated strongest neutralization against BA.4/5, with 50% neutralization titer (NT50) values reaching 174.2; however, neutralization was reduced up to 11-fold, compared to wild-type virus. Both BA.1 convalescent and vaccinated nonconvalescent cohorts displayed the weakest neutralization against BA.4/5, with NT50 values being reduced to 4.6, accompanied by lower numbers of positive neutralizers. Additionally, salivary neutralization against wild-type virus was strongest in vaccinated and BA.2 convalescent subjects, but this elevated neutralization efficiency was lost when challenged with BA.4/5. Our data support the contention that current coronavirus disease 2019 (COVID-19) vaccines efficiently induce humoral immunity. However, antiviral effectiveness in serum and saliva is greatly reduced against novel variants of concern. These results suggest an adjustment of current vaccine strategies to an adapted or alternative vaccine delivery, such as mucosal booster vaccinations, which might establish enhanced or even sterilizing immunity against novel SARS-CoV-2 variants. IMPORTANCE Rising incidences of breakthrough infections caused by SARS-CoV-2 Omicron BA.4/5 have been observed. Although various studies were conducted investigating neutralizing antibodies in sera, mucosal immunity was barely evaluated. Here, we investigated mucosal immunity, since the presence of neutralizing antibodies at mucosal entry sites plays a fundamental role in disease limitation. We found strong induction of serum IgG/IgA, salivary IgA, and neutralization against SARS-CoV-2 wild-type virus in vaccinated/convalescent subjects but detected 10-fold reduced (albeit positive) serum neutralization against BA.4/5. Interestingly, vaccinated and BA.2 convalescent patients demonstrated the greatest serum neutralization against BA.4/5, but this advantageous neutralizing effect was not observed in the saliva. Our data support the contention that current COVID-19 vaccines are very efficient against severe/critical disease progression. Moreover, these results suggest an adjustment of the current vaccine strategy to adapted and alternative vaccine delivery, such as mucosal booster vaccinations, to establish robust sterilizing immunity against novel SARS-CoV-2 variants.

Development of reassortant influenza vaccines: classical reassortment or reverse genetics?

An important feature of influenza vaccines, which distinguishes them from other immunobiological preparations, is that they have no fixed composition. Due to the constant influenza virus antigenic variability, production facilities require timely supply with relevant vaccine strains undoable due to the lack of proper method for the convenient, rapid and uninterrupted development of vaccine strains. Among the licensed influenza vaccines, classical inactivated and live influenza vaccines hold a special place. They are based on reassortant vaccine strains obtained by crossing currently circulating influenza virus with the so-called donor strain (cold-adapted attenuation donor for live influenza vaccines or high yield donor for inactivated vaccines). Vaccine strains for licensed live attenuated influenza vaccines are reassortants with the so-called 6:2 genome formula two genes encoding hemagglutinin and neuraminidase (HA and NA) belong to the current epidemic virus, and six genes encoding internal proteins (PB2, PB1, PA, NP, M and NS) to cold-adapted master donor virus. There is a very limited number of donors of attenuation. In Russia, there are cold-adapted viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69; in the USA (MedImmune) there are viruses A/Ann Arbor/6/60ca (H2N2) and B/Ann Arbor/1/66ca. MedImmune produces vaccine strains using reverse genetics technique. For other countries, this approach for obtaining vaccines is limited due to the need to purchase a license from the patent holders. In Russia, genetic manipulations with strains for the seasonal live influenza vaccine are not yet allowed; reassortants for the Russian live influenza vaccine are created only by classical reassortment in embryonated chicken eggs. Vaccine candidates for the inactivated influenza vaccine are prepared by the classical reassortment method, the requirements for them are more flexible and allow to use diverse genes combinations from wild type virus and master donor virus. High-yielding viruses such as A/PR/8/34 (H1N1), A/Texas/1/77 (H3N2), B/Lee/40 and some others are used as donors of internal genes. Unfortunately, the classical reassortment method does not always allow to promptly obtain a reassortant virus with a 6:2 genome formula. This is hindered by a number of reasons, ranging from the unique properties of a certain epidemic virus ending up with the constellation of genes. The reverse genetics method based on plasmids is an alternative approach to create reassortant vaccine strains allowing to reliably and quickly obtain reassortant viruses of a set 6:2 genome formula. However, this method also has certain weaknesses. This review discusses the advantages and disadvantages of development of conventional influenza vaccine candidates by reverse genetics and classical reassortment in developing chick embryos.

Binding affinity improvement analysis of multiple-mutant Omicron on 2019-nCov to human ACE2 by in silico predictions

ContextSince the outbreak of COVID-19 in 2019, the 2019-nCovcoronavirus has appeared diverse mutational characteristics due to its ownflexible conformation. One multiple-mutant strain (Omicron) with surprisinglyinfective activity outburst, and affected the biological activities of currentdrugs and vaccines, making the epidemic significantly difficult to prevent andcontrol, and seriously threaten health around the world. Importunatelyexploration of mutant characteristics for novel coronavirus Omicron can supplystrong theoretical guidance for learning binding mechanism of mutant viruses.What’s more, full acknowledgement of key mutated-residues on Omicron strain canprovide new methodology of the novel pathogenic mechanism to human ACE2receptor, as well as the subsequent vaccine development.MethodsIn this research, 3D structures of 32 single-point mutationsof 2019-nCov were firstly constructed, and 32-sites multiple-mutant Omicronwere finally obtained based one the wild-type virus by homology modeling method.One total number of 33 2019-nCov/ACE2 complex systems were acquired byprotein-protein docking, and optimized by using preliminary molecular dynamicsimulations. Binding free energies between each 2019-nCov mutation system andhuman ACE2 receptor were calculated, and corresponding binding patternsespecially the regions adjacent to mutation site were analyzed. The resultsindicated that one total number of 6 mutated sites on the Omicron strain playedcrucial role in improving binding capacities from 2019-nCov to ACE2 protein.Subsequently, we performed long-term molecular dynamic simulations andprotein-protein binding energy analysis for the selected 6 mutations. 3infected individuals, the mutants T478K, Q493R and G496S with lower bindingenergies -66.36, -67.98 and -67.09 kcal/mol also presents the high infectivity.These findings indicated that the 3 mutations T478K, Q493R and G496S play thecrucial roles in enhancing binding affinity of Omicron to human ACE2 protein.All these results illuminate important theoretical guidance for future virusdetection of the Omicron epidemic, drug research and vaccine development.

Age-specific transmission dynamics under suppression control measures during SARS-CoV-2 Omicron BA.2 epidemic

BackgroundFrom March to June 2022, an Omicron BA.2 epidemic occurred in Shanghai. We aimed to better understand the transmission dynamics and identify age-specific transmission characteristics for the epidemic.MethodsData on COVID-19 cases were collected from the Shanghai Municipal Health Commission during the period from 20th February to 1st June. The effective reproductive number (Rt) and transmission distance between cases were calculated. An age-structured SEIR model with social contact patterns was developed to reconstruct the transmission dynamics and evaluate age-specific transmission characteristics. Least square method was used to calibrate the model. Basic reproduction number (R0) was estimated with next generation matrix.ResultsR0 of Omicron variant was 7.9 (95% CI: 7.4 to 8.4). With strict interventions, Rt had dropped quickly from 3.6 (95% CI: 2.7 to 4.7) on 4th March to below 1 on 18th April. The mean transmission distance of the Omicron epidemic in Shanghai was 13.4 km (95% CI: 11.1 to 15.8 km), which was threefold longer compared with that of epidemic caused by the wild-type virus in Wuhan, China. The model estimated that there would have been a total 870,845 (95% CI: 815,400 to 926,289) cases for the epidemic from 20th February to 15th June, and 27.7% (95% CI: 24.4% to 30.9%) cases would have been unascertained. People aged 50–59 years had the highest transmission risk 0.216 (95% CI: 0.210 to 0.222), and the highest secondary attack rate (47.62%, 95% CI: 38.71% to 56.53%).ConclusionsThe Omicron variant spread more quickly and widely than other variants and resulted in about one third cases unascertained for the recent outbreak in Shanghai. Prioritizing isolation and screening of people aged 40–59 might suppress the epidemic more effectively. Routine surveillance among people aged 40–59 years could also provide insight into the stage of the epidemic and the timely detection of new variants.Trial registrationWe did not involve clinical trial.