Characterization of influenza A viruses with polymorphism in PB2 residues 701 and 702

The 701 and 702 positions of influenza PB2 polymerase subunit are previously shown to have roles on host range. Limited polymorphisms at these two residues are identified in natural isolates, thereby limiting the study of their role in the polymerase. In this study, we generated 31 viable viruses by random mutagenesis at this region, indicating that these positions can tolerate a wide range of amino acids. These mutants demonstrated varying polymerase activities and viral replication rates in mammalian and avian cells. Notably, some mutants displayed enhanced polymerase activity, yet their replication kinetics were comparable to the wild-type virus. Surface electrostatic charge predication on the PB2 structural model revealed that the viral polymerase activity in mammalian cells generally increases as this region becomes more positively charged. One of the mutants (701A/702E) showed much reduced pathogenicity in mice while others had a pathogenicity similar to the wild-type level. Distinct tissue tropisms of the PB2-701/702 mutants were observed in infected chicken embryos. Overall, this study demonstrates that the PB2-701/702 region has a high degree of sequence plasticity and sequence changes in this region can alter virus phenotypes in vitro and in vivo.published_or_final_versio

Preexisting Antibody-Dependent Cellular Cytotoxicity–Activating Antibody Responses Are Stable Longitudinally and Cross-reactive Responses Are Not Boosted by Recent Influenza Exposure

Cross-reactive influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-activating antibodies are readily detected in healthy adults. However, little is known about the kinetics of these ADCC responses. We used retrospective serial blood samples from healthy donors to investigate this topic. All donors had ADCC responses against 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) and avian influenza A(H7N9) virus hemagglutinins (HAs) despite being seronegative for these viruses in standard hemagglutination inhibition and microneutralization serological assays. A(H1N1)pdm09 exposure did not boost ADCC responses specific for H7 HA antigens. H7 HA ADCC responses were variable longitudinally within donors, suggesting that these cross-reactive antibodies are unstable. We found no correlation between ADCC responses to the H7 HA and either influenza virus-specific immunoglobulin G1 concentration or age

Longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in Saudi Arabia, 2014–2015

Two herds of dromedary camels were longitudinally sampled with nasal and rectal swabs and serum, between September 2014 and May 2015, and the samples were tested for Middle East Respiratory Syndrome (MERS) coronavirus RNA and antibodies. Evidence of MERS-CoV infection was confirmed in one herd on the basis of detection of virus RNA in nasal swabs from three camels and significant increases in the antibody titers from three others. The three viruses were genetically identical, thus indicating introduction of a single virus into this herd. There was evidence of reinfection of camels that were previously seropositive, thus suggesting that prior infection does not provide complete immunity from reinfection, a finding that is relevant to camel vaccination strategies as a means to prevent zoonotic transmission.published_or_final_versio

MERS-CoV in Arabian camels in Africa and Central Asia

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causing infections in humans is genetically indistinguishable from the virus found in Arabian camels (dromedaries) in the Middle East. Although no primary human case of MERS was reported outside the Arabian Peninsula, camel populations in Africa are known to have high prevalence of antibodies against MERS-CoV. We carried out surveillance for MERS-CoV in dromedaries in Africa and Central Asia. By MERS-CoV spike pseudoparticle neutralization assay we confirmed that camel serum samples from African countries have high prevalence of MERS-CoV antibodies. Using RT-qPCR we detected MERS-CoV positives in camel nasal swabs from all different African countries from which samples were collected. However, dromedary serum and swab samples from Kazakhstan in Central Asia were negative for MERS-CoV by these assays. Phylogenetic analysis of the spike gene revealed that MERS-CoVs from Africa formed a cluster closely related to but distinct from the viruses from the Arabian Peninsula. Results from this study suggest that MERS-CoV is actively circulating in dromedary populations in Africa and the virus in Africa is phylogenetically distinct from that in the Middle East.published_or_final_versio

MERS-CoV antibody responses 1 Year after symptom onset, South Korea, 2015

We investigated the kinetics of the Middle East respiratory syndrome coronavirus (MERS-CoV) neutralizing and spike protein antibody titers over the course of 1 year in 11 patients who were confirmed by reverse transcription PCR to have been infected during the outbreak in South Korea in 2015. Robust antibody responses were detected in all survivors who had severe disease; responses remained detectable, albeit with some waning, for <1 year. The duration of viral RNA detection (but not viral load) in sputum significantly correlated with the antibody response magnitude. The MERS S1 ELISA antibody titers correlated well with the neutralizing antibody response. Antibody titers in 4 of 6 patients who had mild illness were undetectable even though most had evidence of pneumonia. This finding implies that MERS-CoV seroepidemiologic studies markedly underestimate the extent of mild and asymptomatic infection. Obtaining convalescent-phase plasma with high antibody titers to treat MERS will be challenging.published_or_final_versio

Stalking influenza by vaccination with pre-fusion headless HA mini-stem for broadly reactive antibodies

Oral Abstract Session: Virology & Pathogenesis: Abstract #O-109Background: Influenza vaccine has been available for over 70 years, yet influenza still causes epidemics or pandemic with substantial morbidity and mortality. The protective responses induced by current human influenza vaccines still primarily depend on vaccine-induced neutralizing antibodies (nAbs) against the HA head. However, the continually evolving influenza virus evades herd immunity induced through natural infection and vaccination by means of antigenic drift and shift. These antigenic drift and shift events render vaccine stockpiling unviable in case of an outbreak or pandemic. In addition, a major shortcoming of current influenza vaccines is its long production time because of existing egg-based or cell-based vaccine manufactory pipelines. Thus, these factors combined necessitate the development of novel influenza vaccine with increased breadth of protection and potential for rapid production and deployment. Method: Recent studies indicated the conserved stem domain contains a greater proportion of vulnerable sites targeted by broadly neutralizing antibodies. Importantly, anti-stem broadly neutralizing antibodies are detectable in some individuals at a low level, suggesting they can be induced naturally by infection and optimized by vaccination. Here, we reported the design of a bacterially expressed polypeptide that mimics a H5 HA stem (i.e. group 1 HA) in the pre-fusion conformation by protein minimization. Results: The absent of HA head domain of this protein could focus host’s antibody responses toward the HA stem. The HA mini-stem folded as a trimer and it was resistant to thermal/chemical stress. It bound to various broadly neutralizing HA stem-specific antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems were protected from morbidity and mortality against lethal challenge by group 1 and group 2 influenza viruses, the first report of cross-group protection. Vaccine-induced antibodies showed broad HA reactivity and no antibody-dependent enhancement activity. Protection from lethal infection was attributed to a broadly reactive antibody response that was able to provide passive protection. Conclusion: The HA mini-stem vaccination can elicit cross-reactive antibody responses that confer robust protection against lethal heterologous influenza A virus challenge from both group 1 and 2 viruses. The recombinant protein is highly stable at room temperature and it can be readily produced in large scale. Our study provides a promising foundation for developing a HA stem-based ‘universal’ influenza vaccine

Potential genetic polymorphism of PB2-701 and 702: implication in virus-host interaction of influenza A virus

Poster Sessions: no. P-152BACKGROUND: PB2-701 of influenza A virus is known as a genetic marker for host adaptation. A D701N mutation of an avian influenza virus can enhance the polymerase activity and viral replication in mammalian cells. In addition, PB2-702 shows host specificity, with most human viruses carry an arginine and most avian viruses carry a lysine at this residue. However, limited polymorphisms at these two residues are found in the natural isolates, limiting the study of their role in the polymerase. METHOD: In order to further elucidate the role of PB2-701/702 in viral fitness and host adaptation, we aim to investigate the potential genetic polymorphism of the PB2-701 and 702 residues by site-directed random mutagenesis of the PB2 gene of the influenza virus in mammalian and avian cells. The polymerase activity, viral replication and pathogenicity of the mutant viruses generated were characterized. RESULTS: A wide range of mutant viruses with different PB2-701 and 702 mutations were successfully isolated, showing that viable viruses with polymorphisms other than PB2-701D/N and 702K/R could be generated. These mutant viruses showed variable polymerase activity in mammalian and avian cells. Several mutants showed enhanced polymerase activity in mammalian cells and comparable viral replication and pathogenicity when compared to the wild-type virus. The variation in the polymerase activity in mammalian cells may be due to the change in net surface charge of the loop around residues 700 – 703 of the PB2 C-terminal when mutations at PB2-701/702 occur. The polymerase activity in mammalian cells generally increases as the surface of the PB2-700 – 703 region becomes more positively charged. On the other hand, some PB2-701/702 mutants (e.g. 701A/702E and 701S/702F) showed reduced polymerase activity and viral replication in mammalian cells. One of them (701A/702E) also showed lower pathogenicity in mice. Importin-α4 was found to have a role in the reduction of the polymerase activity and viral replication of these mutants. Knocking-down the importin-α4 of the mammalian cells enhanced the polymerase activity and/or viral replication of these mutant viruses at 37°C, while the importin-α4 inhibitory effect is not significant in other mutant viruses with high polymerase activity. CONCLUSION: This study demonstrated the potential genetic polymorphism at the residues 701 and 702 of PB2. Some mutant viruses have enhanced polymerase activity when compared to the wild-type, while some have reduced polymerase activity, viral replication and pathogenicity. We also demonstrated that importin-α4 may be responsible for the inhibitory effect observed. Overall, this study may shed light on the study of the role of PB2-701/702 in virus-host interaction

Potential genetic polymorphism of PB2-701 and 702: Implication in virus-host interaction of influenza A virus

Poster presentation - Influenza: no. P45Introduction: PB2-701 of influenza A virus is a genetic marker for host adaptation. A D701N mutation of an avian virus can enhance the polymerase activity and viral replication in mammalian cells. PB2-702 shows host specificity, with most human viruses carry an arginine and most avian viruses carry a lysine. However, limited polymorphisms at these two residues are found in the natural isolates, limiting the study of their role in the polymerase. Objectives: We aim to investigate the potential genetic polymorphism of the PB2-701 and 702 residues by site-directed random mutagenesis of the PB2 gene of the virus. Methods: To elucidate the role of PB2-701/702 in viral fitness, site-directed random mutagenesis of the PB2 gene was performed to generate recombinant viruses with random mutations at PB2-701/702 in mammalian and avian cells. The polymerase activity, viral replication and pathogenicity of the mutant viruses generated were characterized. Results: A wide range of viruses with different PB2-701/702 mutations were isolated. Several mutants demonstrated enhanced polymerase activity in mammalian cells and comparable viral replication and pathogenicity in mice when compared to the wild-type virus. Surface electrostatic charge prediction on the PB2 structural model revealed that the polymerase activity in mammalian cells generally increases as the surface of the PB2-700–703 region becomes more positively charged. On the other hand, some mutants had reduced polymerase activity and viral replication in mammalian cells. One of them (701A/702E) also had lower pathogenicity in mice. Distinct tissue tropism of the PB2-701/702 mutants was observed in chicken embryos. It was found that importin-α4 has a role in the reduction of the polymerase activity in some mutants. Knocking-down the importin-α4 of the mammalian cells enhanced the polymerase activity of the mutants with low polymerase activity, while the importin-α4 inhibitory effect is not significant in other mutants with high polymerase activity. Conclusion: Overall, this study demonstrated the potential genetic polymorphisms at PB2-701/702 and revealed the potential role of importin-α4 in modulating the polymerase activity. The findings in this study may lead to the further study of the role of PB2-701/702 in virus-host interaction and contribute to the control of influenza

Coronavirus infections in horses in Saudi Arabia and Oman

Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT‐PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT‐PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross‐reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections