Poliomyelitis in Intraspinally Inoculated Poliovirus Receptor Transgenic Mice

AbstractMice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice

Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Identification and Characterization of Severe Acute Respiratory Syndrome Coronavirus Replicase Proteins

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis

Evaluation of Replication and Pathogenicity of Avian Influenza A H7 Subtype Viruses in a Mouse Model▿

Avian influenza A H7 subtype viruses pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and, sometimes, death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. Ten H7 viruses were selected for possible vaccine development on the basis of their phylogenetic relationships and geographical locations. The virulence of the 10 viruses for mice and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of antibodies. Most of the viruses showed similar patterns of cross-reactivity with mouse and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages, but the converse was not true. A subset of the viruses was also evaluated for the ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. On the basis of phylogenetic data, induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and their ability to replicate in mice, we have selected A/Netherlands/219/03 (subtype H7N7) and A/chicken/BC/CN-7/04 (subtype H7N3) viruses for vaccine development. The mouse model can be used for the preclinical evaluation of these vaccines against H7 subtype viruses

Prior Infection and Passive Transfer of Neutralizing Antibody Prevent Replication of Severe Acute Respiratory Syndrome Coronavirus in the Respiratory Tract of Mice

Following intranasal administration, the severe acute respiratory syndrome (SARS) coronavirus replicated to high titers in the respiratory tracts of BALB/c mice. Peak replication was seen in the absence of disease on day 1 or 2, depending on the dose administered, and the virus was cleared within a week. Viral antigen and nucleic acid were detected in bronchiolar epithelial cells during peak viral replication. Mice developed a neutralizing antibody response and were protected from reinfection 28 days following primary infection. Passive transfer of immune serum to naïve mice prevented virus replication in the lower respiratory tract following intranasal challenge. Thus, antibodies, acting alone, can prevent replication of the SARS coronavirus in the lung, a promising observation for the development of vaccines, immunotherapy, and immunoprophylaxis regimens

Mucosal Immunization of Rhesus Monkeys against Respiratory Syncytial Virus Subgroups A and B and Human Parainfluenza Virus Type 3 by Using a Live cDNA-Derived Vaccine Based on a Host Range-Attenuated Bovine Parainfluenza Virus Type 3 Vector Backbone

Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide