Wild-type Virus Research Articles

A Historical Perspective on Respiratory Syncytial Virus Prevention: A Journey Spanning Over Half a Century From the Setback of an Inactive Vaccine Candidate to the Success of Passive Immunization Strategy.

The efforts to prevent respiratory syncytial virus (RSV) infection in infants span over half a century. RSV vaccine development began in the 1960s, and it confronted a significant disappointment after testing a formalin-inactivated RSV (FI RSV) vaccine candidate. This inactivated RSV vaccine was not protective. A large number of the vaccinated RSV-naive children, when subsequently exposed to natural RSV infection from wild-type virus in the community, developed severe lung inflammation termed enhanced respiratory disease. This resulted in a halt in RSV vaccine development. In the 1990s, attention turned to the potential for passive protection against severe RSV disease with immunoglobulin administration. This led to studies on using standard intravenous immunoglobulins in high-risk infants, followed by high-titer RSV immunoglobulin preparation and, subsequently, the development of RSV monoclonal antibodies. Over the past 25 years, palivizumab has been recognized as a safe and effective monoclonal antibody as a prevention strategy for RSV in high-risk children. Its high cost and need for monthly administration, however, has hindered its use to ~2% of the birth cohort, neglecting the vast majority of newborns, including healthy full-term infants who comprise the largest portion of RSV hospitalizations and the greatest part of the burden of RSV disease. Still these efforts, helped pave the way for the present advances in RSV prevention that hold promise for mitigating severe RSV disease for all infants.

COVID-19 admissions: Trying to define the real impact of infection in hospitalized patients

Introduction and objectiveSeveral studies have suggested that the hospitalization rate for COVID-19 in children and adolescents may reflect the prevalence of the infection rather than the severity of the disease. The aim of this study was to describe the clinical features of hospitalised paediatric patients with SARS-CoV-2 infection in order to understand if the infection was the reason for admission. MethodsRetrospective cohort study including patients aged 0–18 years with SARS-CoV-2 infection or multisystem inflammatory syndrome in children (MIS-C) admitted to a tertiary care children’s hospital in Spain between 01/01/2020 and 12/31/2021. Results228 patients were included, corresponding to 150 cases of COVID-related admission (SARS-CoV-2 infection as main cause of hospitalization) and 78 of non-COVID-related admission (SARS-CoV-2 infection unrelated to the hospitalization).In the group of COVID-related admissions, 58 patients had comorbidities. Forty-nine patients had acute respiratory disease (pneumonia, bronchospasm or bronchiolitis). Multisystem inflammatory syndrome in children was diagnosed in 27 and was significantly more frequent in the first year of the pandemic (wild type virus). Eighty percent of patients with acute respiratory disease needed respiratory support, mostly low-flow oxygen therapy. The severity of the disease was similar in all virus variants. Two patients (both with severe comorbidities) died from COVID-related conditions. ConclusionsIn our study, one third of the patients were admitted with SARS-CoV-2 infection but not because of it. Acute respiratory disease was less frequent and had a better prognosis compared to the adult population, while MIS-C was a major cause of morbidity and hospitalization. The fatality rate was extremely low.

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

Development of Live Vaccine Candidates for Canine Influenza H3N2 Using Naturally Truncated NS1 Gene

The NS1 influenza protein of influenza A virus is a viral nonstructural protein encoded by the NS gene segment that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced protein. We isolated an equine influenza virus with a naturally truncated NS1 gene in our previous study. In this current research, we inserted this partially truncated NS gene into the H3N2 canine influenza virus using reverse genetics to develop a live attenuated vaccine strain. To evaluate whether the developed strain is suitable as a live vaccine candidate, we compared its replication kinetics with wild-type virus in MDCK cells and specific pathogen-free eggs. Additionally, we investigated host antiviral gene expression, viral replication in the respiratory system, and associated lung tissue damage in mice experiments. To confirm the efficacy of the vaccine candidate, we evaluated the immunogenicity and protectivity of the developed vaccine strain against canine influenza H3N2, compared with a commercial inactivated vaccine. Through these experiments, it was confirmed that the naturally truncated NS1 inserted virus has sufficient potential as a live vaccine candidate, and we hopefully expect that this study would make a great contribution to the development of a live vaccine for canine influenza H3N2.

Evaluation of humoral immune response after yellow fever infection: an observational study on patients from the 2017-2018 sylvatic outbreak in Brazil.

Between 2016 and 2018, Brazil experienced major sylvatic yellow fever (YF) outbreaks that caused hundreds of casualties, with Minas Gerais (MG) being the most affected state. These outbreaks provided a unique opportunity to assess the immune response triggered by the wild-type (WT) yellow fever virus (YFV) in humans. The plaque reduction neutralization test (PRNT) is currently the standard method to assess the humoral immune response to YFV by measuring neutralizing antibodies (nAbs). The present study aimed to evaluate the humoral immune response of patients from the 2017-2018 sylvatic YF outbreak in MG with different disease outcomes by using PRNTs with a WT YFV strain, isolated from the 2017-2018 outbreak, and a vaccine YFV strain. Samples from naturally infected YF patients were tested, in comparison with healthy vaccinees. Results showed that both groups presented different levels of nAb against the WT and vaccine strains, and the levels of neutralization against the strains varied homotypically and heterotypically. Results based on the geometric mean titers (GMTs) suggest that the humoral immune response after a natural infection of YFV can reach higher levels than that induced by vaccination (GMT of patients against WT YFV compared to GMT of vaccinees, P < 0.0001). These findings suggest that the humoral immune responses triggered by the vaccine and WT strains of YFV are different, possibly due to genetic and antigenic differences between these viruses. Therefore, current means of assessing the immune response in naturally infected YF individuals and immunological surveillance methods in areas with intense viral circulation may need to be updated.IMPORTANCEYellow fever is a deadly febrile disease caused by the YFV. Despite the existence of effective vaccines, this disease still represents a public health concern worldwide. Much is known about the immune response against the vaccine strains of the YFV, but recent studies have shown that it differs from that induced by WT strains. The extent of this difference and the mechanisms behind it are still unclear. Thus, studies aimed to better understand the immune response against this virus are relevant and necessary. The present study evaluated levels of neutralizing antibodies of yellow fever patients from recent outbreaks in Brazil, in comparison with healthy vaccinees, using plaque reduction neutralization tests with WT and vaccine YFV strains. Results showed that the humoral immune response in naturally infected patients was higher than that induced by vaccination, thus providing new insights into the immune response triggered against these viruses.

Cross-neutralizing antibody against emerging Omicron subvariants of SARS-CoV-2 in infection-naïve individuals with homologous BNT162b2 or BNT162b2(WT + BA.4/5) bivalent booster vaccination

Since the emergence of SARS-CoV-2, different variants and subvariants successively emerged to dominate global virus circulation as a result of immune evasion, replication fitness or both. COVID-19 vaccines continue to be updated in response to the emergence of antigenically divergent viruses, the first being the bivalent RNA vaccines that encodes for both the Wuhan-like and Omicron BA.5 subvariant spike proteins. Repeated infections and vaccine breakthrough infections have led to complex immune landscapes in populations making it increasingly difficult to assess the intrinsic neutralizing antibody responses elicited by the vaccines. Hong Kong’s intensive COVID-19 containment policy through 2020–2021 permitted us to identify sera from a small number of infection-naïve individuals who received 3 doses of the RNA BNT162b2 vaccine encoding the Wuhan-like spike (WT) and were boosted with a fourth dose of the WT vaccine or the bivalent WT and BA.4/5 spike (WT + BA.4/5). While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 (WT + BA.4/BA.5) bivalent vaccine elicited significantly higher plaque neutralizing antibodies to Omicron subvariants BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1, compared to BNT162b2 monovalent vaccine. The single amino acid substitution that differentiates the spike of JN.1 from BA.2.86 resulted in a profound antigenic change.

No two without three: Modeling dynamics of the trio RNA virus-defective interfering genomes-satellite RNAs

Almost all viruses, regardless of their genomic material, produce defective viral genomes (DVG) as an unavoidable byproduct of their error-prone replication. Defective interfering (DI) elements are a subgroup of DVGs that have been shown to interfere with the replication of the wild-type (WT) virus. Along with DIs, other genetic elements known as satellite RNAs (satRNAs), that show no genetic relatedness with the WT virus, can co-infect cells with WT helper viruses and take advantage of viral proteins for their own benefit. These satRNAs have effects that range from reduced symptom severity to enhanced virulence. The interference dynamics of DIs over WT viruses has been thoroughly modeled at within-cell, within-host, and population levels. However, nothing is known about the dynamics resulting from the nonlinear interactions between WT viruses and DIs in the presence of satellites, a process that is frequently seen in plant RNA viruses and in biomedically relevant pathosystems like hepatitis B virus and its δ satellite. Here, we look into a simple phenomenological mathematical model that describes how a WT virus replicates and produces DIs in presence of a satRNA. The WT virus is subject to mechanisms of complementation, competition, and various levels of interference from DIs and the satRNA. Examining the dynamics analytically and numerically reveals three possible regimes: (i) full extinction, (ii) satellite extinction and virus-DIs coexistence and (iii) full coexistence. Assuming DIs replicate faster than the satRNA owed to their smaller size drives to scenario (ii), which implies that DIs could wipe out the satRNA. In addition, a small region of the parameter space exists wherein the system is bistable (either scenarios (ii) or (iii) are concurrently stable). We have identified transcritical bifurcations in the transitions between scenarios (i) to (iii) and saddle–node bifurcations behind the change from bistability to monostability. Despite the model simplicity, our findings may have applications in biomedicine and agronomy. They will cast light on the dynamics of this three-species system and aid in the identification of scenarios in which the clearance of the satRNAs may be possible thus e.g., allowing for less severe disease symptoms.

Detection of SARS-CoV-2 variants distributed from March 2020 to May 2022 and their effect on the infection severity in Iraqi population

New SARS-CoV-2 variants appeared in late December 2020 as Mutations accumulated in the original virus. This study aimed to provide a local database about variants of COVID-19 circulating in the Iraqi population from 2020 to 2022 and the time of emergence of new strains each month since very few local studies have documented its existence in the country. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays were employed to 319 collected and analyzed nasal swabs to determine whether an infection had occurred. A sophisticated diagnostic kit that sorted the distinctive mutations was implemented to evaluate the variants. Results showed Younger patients were more likely to be infected with the Alpha variant (66 patients) than older people (43 patients). Additionally, patients with wild-type infestations had more robust viral load and lower Ct threshold values, culminating in an increase in severity during infection with wild-type virus 26/32 (81.250). Meanwhile, 65/109(59.63%) of patients infected with the Alpha variant developed severe and critical illness and 51/84(60.71%)were infected with Delta or Delta plus variants. In conclusion, the Alpha variant had the highest infection percentage of 109(46.6%), followed by Delta or Delta plus variant 84(26.33%), Beta or Gamma variants 47(20.1%), Omicron variant 46(19.6%), and finally wild-type virus of 32(13.7%). February 2020 witnessed a preliminary finding of the wild-type, while the Alpha variant emerged in December 2020, Beta/Gamma variances were recognized in December 2020, Delta/Delta plus variances began in April 2021, and the Omicron variant debuted in March 2022. Keywords: SARS-CoV-2, Mutation, rRT-PCR, Coronavirus disease 2019, TaqPath, cycle threshold (Ct) value

Detection of SARS-CoV-2 variants distributed from March 2020 to May 2022 and their effect on the infection severity in Iraqi population

New SARS-CoV-2 variants appeared in late December 2020 as Mutations accumulated in the original virus. This study aimed to provide a local database about variants of COVID-19 circulating in the Iraqi population from 2020 to 2022 and the time of emergence of new strains each month since very few local studies have documented its existence in the country. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays were employed to 319 collected and analyzed nasal swabs to determine whether an infection had occurred. A sophisticated diagnostic kit that sorted the distinctive mutations was implemented to evaluate the variants. Results showed Younger patients were more likely to be infected with the Alpha variant (66 patients) than older people (43 patients). Additionally, patients with wild-type infestations had more robust viral load and lower Ct threshold values, culminating in an increase in severity during infection with wild-type virus 26/32 (81.250). Meanwhile, 65/109(59.63%) of patients infected with the Alpha variant developed severe and critical illness and 51/84(60.71%)were infected with Delta or Delta plus variants. In conclusion, the Alpha variant had the highest infection percentage of 109(46.6%), followed by Delta or Delta plus variant 84(26.33%), Beta or Gamma variants 47(20.1%), Omicron variant 46(19.6%), and finally wild-type virus of 32(13.7%). February 2020 witnessed a preliminary finding of the wild-type, while the Alpha variant emerged in December 2020, Beta/Gamma variances were recognized in December 2020, Delta/Delta plus variances began in April 2021, and the Omicron variant debuted in March 2022. Keywords: SARS-CoV-2, Mutation, rRT-PCR, Coronavirus disease 2019, TaqPath, cycle threshold (Ct) value

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.

Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

Ingresos COVID-19: intentando comprender el impacto real de la infección en pacientes hospitalizados

Introduction and objectiveSeveral studies have suggested that the hospitalization rate for COVID-19 in children and adolescents may reflect the prevalence of the infection rather than the severity of the disease. The aim of this study was to describe the clinical features of hospitalised paediatric patients with SARS-CoV-2 infection in order to understand if the infection was the reason for admission. MethodsRetrospective cohort study including patients aged 0 to 18 years with SARS-CoV-2 infection or multisystem inflammatory syndrome in children (MIS-C) admitted to a tertiary care children's hospital in Spain between 01/01/2020 and 12/31/2021. Results228 patients were included, corresponding to 150 cases of COVID-related admission (SARS-CoV-2 infection as main cause of hospitalization) and 78 of non-COVID-related admission (SARS-CoV-2 infection unrelated to the hospitalization).In the group of COVID-related admissions, 58 patients had comorbidities. Forty-nine patients had acute respiratory disease (pneumonia, bronchospasm or bronchiolitis). Multisystem inflammatory syndrome in children was diagnosed in 27 and was significantly more frequent in the first year of the pandemic (wild type virus). Eighty percent of patients with acute respiratory disease needed respiratory support, mostly low-flow oxygen therapy. The severity of the disease was similar in all virus variants. Two patients (both with severe comorbidities) died from COVID-related conditions. ConclusionsIn our study, one third of the patients were admitted with SARS-CoV-2 infection but not becauseof it. Acute respiratory disease was less frequent and had a better prognosis compared to the adult population, while MIS-C was a major cause of morbidity and hospitalization. The fatality rate was extremely low.

Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.

Immunogenicity and safety of COVID-19 booster vaccination: A population-based clinical trial to identify the best vaccination strategy

BackgroundVarious SARS-CoV-2 variants of concerns (VOCs) characterized by higher transmissibility and immune evasion have emerged. Despite reduced vaccine efficacy against VOCs, currently available vaccines provide protection. Population-based evidence on the humoral immune response after booster vaccination is crucial to guide future vaccination strategies and in preparation for imminent COVID-19 waves. MethodsThis multicenter, population-based cohort study included 4697 individuals ≥18 years of age who received a booster vaccination. Antibody levels against SARS-CoV-2 receptor binding domain (RBD) and neutralizing antibodies against wild-type (WT) virus and Omicron variants were assessed at baseline (day of booster vaccination) and after four weeks. Safety was evaluated daily within the first week using a participant-completed electronic diary. Antibody levels were compared across different vaccination strategies, taking into account individual host factors. ResultsOur main model including 3838 participants revealed that individuals who received a booster with mRNA-1273 compared to BNT162b2 vaccine had a significantly higher increase (95 %CI) in anti-RBD-antibody levels (37,707 BAU/mL [34,575–40,839] vs. 27,176 BAU/mL [26,265–28,087]), and of neutralization levels against WT (1,681 [1490–1872] vs. 1141 [1004–1278] and Omicron variant (422 [369–474] vs. 329 [284–374]). Neutralizing antibody titres highly correlated with anti-RBD antibodies, with neutralizing capacity 4.4 fold higher against WT compared to Omicron. No differences in safety were found between the two booster vaccines. ConclusionOur study underlines the superiority of a booster vaccination with mRNA-1273, independent of the primary vaccination and therefore provides guidance on the vaccination strategy.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.