Microneutralization Test Research Articles

Development of indigenous IgG ELISA for the detection of anti-SARS-CoV-2 IgG.

Background & objectives:Since the beginning of the year 2020, the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted humankind adversely in almost all spheres of life. The virus belongs to the genus Betacoronavirus of the family Coronaviridae. SARS-CoV-2 causes the disease known as coronavirus disease 2019 (COVID-19) with mild-to-severe respiratory illness. The currently available diagnostic tools for the diagnosis of COVID-19 are mainly based on molecular assays. Real-time reverse transcription-polymerase chain reaction is the only diagnostic method currently recommended by the World Health Organization for COVID-19. With the rapid spread of SARS-CoV-2, it is necessary to utilize other tests, which would determine the burden of the disease as well as the spread of the outbreak. Considering the need for the development of such a screening test, an attempt was made to develop and evaluate an IgG-based ELISA for COVID-19.Methods:A total of 513 blood samples (131 positive, 382 negative for SARS-CoV-2) were collected and tested by microneutralization test (MNT). Antigen stock of SARS-CoV-2 was prepared by propagating the virus in Vero CCL-81 cells. An IgG capture ELISA was developed for serological detection of anti-SARS-CoV-2 IgG in serum samples. The end point cut-off values were determined by using receiver operating characteristic (ROC) curve. Inter-assay variability was determined.Results:The developed ELISA was found to be 92.37 per cent sensitive, 97.9 per cent specific, robust and reproducible. The positive and negative predictive values were 94.44 and 98.14 per cent, respectively.Interpretation & conclusions:This indigenously developed IgG ELISA was found to be sensitive and specific for the detection of anti-SARS-CoV-2 IgG in human serum samples. This assay may be used for determining seroprevalence of SARS-CoV-2 in a population exposed to the virus.

Zika Virus in West Africa: A Seroepidemiological Study between 2007 and 2012

Background: Before its recent spread, serological studies conducted between the 1960s and the 1990s showed the wide presence of Zika virus in Africa, but in recent years, limited data are available on its circulation in the African population. The aim of this study was to evaluate the immunity to Zika virus in a selected cohort of healthy subjects from West Africa between 2007 and 2012. Methods: Human serum samples were collected in 2007 and in 2011/2012 from a cohort of 2-29-year-old subjects from Mali, Senegal, and The Gambia. A sample that tested positive by Zika virus IgG ELISA and by Zika virus microneutralization test was defined as positive. Findings: In 2007, the highest seroprevalence to Zika was 21·9% (95%CI 14·08-31·47), found in Senegal among 18-29-year-old subjects. In 2011/2012, the highest seroprevalence, 22·7% (95%CI 13·79-33·79), was found still in Senegal, but in 11-17-year-old subjects. During the two study periods the lowest prevalence was found in Mali, where few positive cases were found and only in 18-29-year-old subjects. The Gambia showed an intermediate seroprevalence. In the three countries, seroprevalence to Zika virus was strongly associated with increasing age. Interpretation: This study contributes to understanding Zika virus circulation within three different ecological and demographic contexts with scarce or no data currently available. Results showed that Zika virus circulated actively in West Africa between 2007 and 2011/2012, but with some geographic specificity. As seropositivity to Zika virus increases with age, women are at high risk to be infected during their reproductive age. Funding Statement: None. Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: The present study was approved by Ethic Committees in Mali, Senegal and The Gambia.

Exposure of Wild Ungulates to the Usutu and Tick-Borne Encephalitis Viruses in France in 2009-2014: Evidence of Undetected Flavivirus Circulation a Decade Ago.

Flaviviruses have become increasingly important pathogens in Europe over the past few decades. A better understanding of the spatiotemporal distribution of flaviviruses in France is needed to better define risk areas and to gain knowledge of the dynamics of virus transmission cycles. Serum samples from 1014 wild boar and 758 roe deer from 16 departments (administrative units) in France collected from 2009 to 2014 were screened for flavivirus antibodies using a competitive ELISA (cELISA) technique. Serum samples found to be positive or doubtful by cELISA were then tested for antibodies directed against West Nile virus (WNV), Usutu virus (USUV), Bagaza virus (BAGV), and tick-borne encephalitis/Louping ill viruses (TBEV/LIV) by microsphere immunoassays (except BAGV) and micro-neutralization tests. USUV antibodies were detected only in southeastern and southwestern areas. TBEV/LIV antibodies were detected in serum samples from eastern, southwestern and northern departments. The results indicate continuous circulation of USUV in southern France from 2009 to 2014, which was unnoticed by the French monitoring system for bird mortality. The findings also confirm wider distribution of TBEV in the eastern part of the country than of human clinical cases. However, further studies are needed to determine the tick-borne flavivirus responsible for the seroconversion in southwestern and northern France.

Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic coronavirus that was identified in 2012. MERS-CoV infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. The MERS-CoV spike (S) protein is a major target for neutralizing antibodies in infected patients. The MERS-CoV microneutralization test (MNt) is the gold standard method for demonstrating prior infection. However, this method requires the use of live MERS-CoV in biosafety level 3 (BSL-3) containment. The present work describes the generation and validation of S protein-bearing vesicular stomatitis virus (VSV) pseudotype particles (VSV-MERS-CoV-S) in which the VSV glycoprotein G gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect MERS-CoV neutralizing antibodies under BSL-2 conditions. Using a panel of human sera from confirmed MERS-CoV patients, the VSV-MERS-CoV particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live MERS-CoV. The results suggest that the VSV-MERS-CoV-S pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect MERS-CoV neutralizing antibodies in human sera.

A systematic review and meta-analysis of cross-reactivity of antibodies induced by H7 influenza vaccine

ABSTRACTInoculation with vaccine is the major intervention currently used to prevent influenza infections. However, it will be a challenge to produce and implement a new vaccine when a novel highly pathogenic influenza virus emerges in humans as significant infections. H7 subtype influenza viruses have similar epitopes on hemagglutinin, which can induce cross-reactive antibodies. In this study, a meta-analysis of the cross-reactivity of antibodies induced by one H7 subtype influenza vaccine against other H7 subtypes was performed. Database search was conducted in PubMed, Cochrane Library, EMBASE, MEDLINE, Chinese Biological Medicine Database (CBM), and Wanfang. A total of 9 articles comprising 811 human subjects were included in this meta-analysis. All assessed H7 influenza vaccines induced vaccine strain-specific protective antibodies [seroconversion rate (SCR) = 0.74, 95% CI (0.65, 0.82); seroprotection rate (SPR) = 0.81, 95% CI (0.78, 0.83)]. All H7 influenza virus monovalent vaccines exhibited cross-reactivity tested by hemagglutinin inhibition test (HI), microneutralization test (MN) and immunosorbent assay (ELISA) to other H7 subtype viruses. H7N1, H7N3, H7N7, and H7N9 vaccines elicited cross-reactive antibodies against other H7 subtype influenza viruses [SCR = 0.66, 95% CI (0.50, 0.82); SPR = 0.79, 95% CI (0.67, 0.91)]. The pooled SCR (95%CI) of cross-reactivity of H7N1 and H7N3 vaccines were 0.88 (0.85, 0.91) and 0.40 (0.26, 0.54), respectively. The consolidated SPR (95%CI) of H7N1 and H7N7 vaccines were 0.89 (0.86, 0.92) and 0.93 (0.81, 1.06). All H7 vaccines induced cross-reactive antibodies against H7N9 viruses [SCR = 0.69, 95% CI (0.52, 0.86); SPR = 0.85, 95% CI (0.76, 0.94)]. H7 vaccines can be used to limit influenza infection when a new highly pathogenic H7 virus appears.

Impact of Zika Virus Emergence in French Guiana: A Large General Population Seroprevalence Survey.

BackgroundSince the identification of Zika virus (ZIKV) in Brazil in May 2015, the virus has spread throughout the Americas. However, ZIKV burden in the general population in affected countries remains unknown.MethodsWe conducted a general population survey in the different communities of French Guiana through individual interviews and serologic survey during June–October 2017. All serum samples were tested for anti-ZIKV immunoglobulin G antibodies using a recombinant antigen-based SGERPAxMap microsphere immunoassay, and some of them were further evaluated through anti-ZIKV microneutralization tests.ResultsThe overall seroprevalence was estimated at 23.3% (95% confidence interval [CI], 20.9%–25.9%) among 2697 participants, varying from 0% to 45.6% according to municipalities. ZIKV circulated in a large majority of French Guiana but not in the most isolated forest areas. The proportion of reported symptomatic Zika infection was estimated at 25.5% (95% CI, 20.3%–31.4%) in individuals who tested positive for ZIKV.ConclusionsThis study described a large-scale representative ZIKV seroprevalence study in South America from the recent 2015–2016 Zika epidemic. Our findings reveal that the majority of the population remains susceptible to ZIKV, which could potentially allow future reintroductions of the virus.

Transmission risk of avian influenza virus along poultry supply chains in Guangdong, China

ObjectivesAvian influenza viruses (AIVs) poise significant risk to human health and the poultry industry. We evaluated the transmission risk along the poultry supply chain. MethodsDuring October 2015 and July 2016, four rounds of cross-sectional surveys were performed to characterize AIV spread in farms, transport vehicles, slaughterhouses, wholesale and retail live poultry markets (LPMs). Poultry cloacal and oral swabs, environmental swabs, bioaerosol samples and human sera were collected. Poultry and environmental samples were tested for AIVs by rRT-PCR, further subtyped by next generation sequencing. Previous human H9N2 infections were identified by hemagglutination inhibition and microneutralization tests. Logistic regression was fitted to compare AIV transmission risk in different settings. ResultsAIVs was detected in 23.9% (424/1771) of the poultry and environmental samples. AIV detection rates in farms, transport vehicles, wholesale and retail LPMs were 4.5%, 11.1%, 30.3% and 51.2%, respectively. 5.2%, 8.3% and 12.8% of the poultry workers were seropositive in farms, wholesale and retail LPMs, respectively. The regression analysis showed that virus detection and transmission risk to human increased progressively along the poultry supply chain. ConclusionsStrengthening control measures at every level along the poultry supply chain, using a one health approach, is crucial to control AIV circulation.

Seasonal influenza vaccines induced high levels of neutralizing cross-reactive antibody responses against different genetic group influenza A(H1N1)pdm09 viruses

Influenza A(H1N1)pdm09 viruses have been circulating throughout the world since the 2009 pandemic. A/California/07/2009 (H1N1) virus was included in seasonal influenza vaccines for seven years altogether, providing a great opportunity to analyse vaccine-induced immunity in relation to the postpandemic evolution of the A(H1N1)pdm09 virus. Serum antibodies against various epidemic strains of influenza A(H1N1)pdm09 viruses were measured among health care workers (HCWs) by haemagglutination inhibition and microneutralization tests before and after 2010 and 2012 seasonal influenza vaccinations. We detected high responses of vaccine-induced neutralizing antibodies to six distinct genetic groups. Our results indicate antigenic similarity between vaccine and circulating A(H1N1)pdm09 strains, and substantial vaccine-induced immunity against circulating epidemic viruses.

Humoral and cellular immunity against both ZIKV and poxvirus is elicited by a two-dose regimen using DNA and non-replicating vaccinia virus-based vaccine candidates

The Zika virus (ZIKV) and poxvirus infection are considered as public health emergencies, necessitating the development of effective vaccines. Here, we report novel recombinant DNA-based and non-replicating vaccinia virus (NTV)-based vaccine candidates that express the precursor membrane-envelope (prME) or envelope (E) glycoproteins of ZIKV. After immunization of BABL/c mice with the vaccines using a homologous protocol (DNA/DNA, NTV/NTV) or heterogeneous (DNA/NTV) protocol, a similar level of anti-E IgG and neutralizing antibodies (microneutralization test) were detected in the mice. However, a significantly higher level of E-specific T cell responses was elicited in mice when a heterogeneous prime-boost protocol was used (DNA/NTV) with either the DNA-based or NTV-based vaccines. Furthermore, neutralizing antibodies and a T cell immune response against the vaccinia virus (VV) were detected in mice that were subjected to the prime-boost protocol (DNA/NTV), whereas those subjected to a homologous NTV/NTV protocol had higher levels of anti-VV IgG and neutralizing antibodies. Lastly, a novel H-2d-restricted CD8 T-cell epitope, VRSYCYEASISDMAS, was identified in the ZIKV E protein. These data demonstrate proof of concept of a bivalent vaccine candidate against ZIKV and orthopoxvirus, and support the use of DNA-prME prime and NTV-E boost protocols to protect against ZIKV and orthopoxvirus infections.

Development of low bio-containment assays to characterise the antibody responses in pigs to Nipah virus vaccine candidates

Nipah virus (NiV) is a zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. Since 1998, NiV outbreaks have occurred in Malaysia, Bangladesh and India. NiV poses a significant pandemic threat due to its broad host range and the widespread distribution of its natural host, the Pteropus fruit bat. Despite this, there are currently no licenced therapeutics or vaccines for use in either humans or livestock. Aiming to develop a safe and inexpensive vaccine to protect livestock in future outbreaks and to prevent onward transmission to humans, we are evaluating the immunogenicity and efficacy of three candidate vaccines in pigs. These vaccines are based on the NiV G or F surface glycoproteins, proteins which are essential for mediating virus-cell or cell-cell entry. The candidate vaccines include: (i) a recombinant soluble NiV G protein subunit, (ii) a recombinant molecular clamp stabilised NiV F protein subunit and (iii) a replication deficient, adenoviral vectored NiV G protein. We have developed low biocontainment assays to characterise the antibody responses to NiV and to aid identification of immune correlates of protection through quantification of both antigen-specific and neutralising antibody responses to our vaccine candidates. This includes anti-NiV F/G indirect ELISAs, a microneutralisation test using pseudotyped particles, and a microfusion inhibition test using a quantifiable cell-cell fusion assay. Using these assays we have demonstrated that all three of our novel vaccines are immunogenic in pigs and capable of generating a robust antibody response, with evidence for neutralising antibodies.

Development of a HA1-specific enzyme-linked immunosorbent assay against pandemic influenza virus A H1N1.

PurposeEnzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein.Materials and MethodsrHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice.ResultsOur ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model.ConclusionAn ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.

Middle East respiratory syndrome coronavirus infection in non-camelid domestic mammals

ABSTRACT Dromedary camels are natural host of the Middle East respiratory syndrome coronavirus (MERS-CoV). However, there are limited studies of MERS-CoV infection of other domestic mammals exposed to infected dromedaries. We expanded our surveillance among camels in Egypt, Tunisia, and Senegal to include other domestic mammalian species in contact with infected camels. A total of 820 sera and 823 nasal swabs from cattle, sheep, goats, donkeys, buffaloes, mules, and horses were collected. Swabs were tested using RT-PCR and virus RNA-positive samples were genetically sequenced and phylogenetically analysed. Sera were screened using virus microneutralization tests and positive sera (where available) were confirmed using plaque reduction neutralization tests (PRNT). We detected 90% PRNT confirmed MERS-CoV antibody in 35 (55.6%) of 63 sera from sheep collected from Senegal, two sheep (1.8%) of 114 in Tunisia and a goat (0.9%) of 107 in Egypt, with titres ranging from 1:80 to ≥1:320. We detected MERS-CoV RNA in swabs from three sheep (1.2%) of 254 and five goats (4.1%) of 121 from Egypt and Senegal, as well as one cow (1.9%) of 53 and three donkeys (7.1%) of 42 from Egypt. Partial sequences of the RT-PCR amplicons confirmed specificity of the results. This study showed that domestic livestock in contact with MERS-CoV infected camels may be at risk of infection. We recommend expanding current MERS-CoV surveillance in animals to include other livestock in close contact with dromedary camels. The segregation of camels from other livestock in farms and live animal markets may need to be considered.

A neuraminidase activity-based microneutralization assay for evaluating antibody responses to influenza H5 and H7 vaccines.

Outbreaks of the highly pathogenic avian influenza H5N1 and H7N9 viruses have spurred an unprecedented research effort to develop antivirals and vaccines against influenza. Standardized methods for vaccine evaluation are critical for facilitating vaccine development. Compared with hemagglutination inhibition assays, mounting evidence suggest that microneutralization tests (MNTs) is a better choice for the evaluation of candidate pandemic influenza vaccines because they measure neutralizing antibody activity in cell cultures and are more sensitive in detecting H5 and H7. Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses. Compared to the conventional microneutralization assay (ELISA-MNT), the NA-MNT is faster with lower intra- and inter-assay variations, while no difference in geometric mean titers was found between these two assays for the evaluation of H5N1 and H7N9 vaccines. These results suggest that NA-MNT is a reliable and high throughput method which could facilitate the development of candidate pandemic influenza vaccine.

West Nile virus infection in horses in Saudi Arabia (in 2013-2015).

West Nile virus (WNV) is an important emerging zoonotic arbovirus giving rise to clinical syndromes of varying severity in humans and horses. Culex mosquitoes are the main vector. Although WNV has been reported in many countries in the Middle East and Asia, little is known about its prevalence in equine populations in the Arabian Peninsula. We have carried out a serological study on 200 horses to assess WNV infection in the Eastern and Central regions of Saudi Arabia in 2013-2015. Sera were tested for the presence of WNV antibodies in parallel using a commercial enzyme-linked immunosorbent assay (ELISA) kit and microneutralization (MN) tests. In comparison with the MN assay used as "gold standard," we find the ELISA had a sensitivity of 94.7% and specificity of 80.1%. The prevalence of WNV neutralizing antibody ranged from 5 (17.3%) of 29 sera collected in Riyadh up to 15 (55.6%) of 27 sera collected from Al-Qateef. These findings highlight the need to be aware of the possibility of WNV disease in humans and horses presenting with central nervous system disease in the Kingdom of Saudi Arabia.

Seroprevalence of Poliomyelitis Antibodies Among Children Aged 1 to 4 Years Old and Factors Associated With Poliovirus Susceptibility; Mexican Health and Nutrition Survey, 2012.

BackgroundAn essential component of the “Polio Eradication and Endgame Strategic Plan 2013–2018” is the evaluation of population immunity. Mexico introduced the inactivated polio vaccine (IPV) into its routine immunization schedule in 2007 but continued to give trivalent oral polio vaccine OPV twice a year during National Health Weeks through 2016.MethodsTo describe the seroprevalence of poliomyelitis among children one to four years old in Mexico and analyze risk factors for susceptibility. We detected antibodies to poliovirus type 1 by microneutralization test in 966 serum samples randomly selected from the National Health and Nutrition Survey, 2012. We assessed variables associated with susceptibility using multivariable logistic regression.ResultsThe overall weighted seroprevalence of the general population was 98.39% (95% confidence interval [CI] 96.76–99.21). We found significant differences of prevalence according to age (94.39%, 95% CI 87.56–97.58; 99.02%, 95% CI 95.68–99.79; 99.82%, 95% CI 98.77–99.98; and 100% among children 1, 2, 3, and 4 years old respectively) and number of IPV doses (96.91%, 95% CI 90.55–99.44; 100%; 97.85%, 95% CI 94.46–99.18; and 99.92%, 95% CI 99.45–99.98 for 1 2, 3, and 4 number of doses, respectively). Multivariate analyses showed that susceptibility was associated with younger age, fewer doses of IPV, and certain socioeconomic levels.ConclusionsOverall seroprevalence was high. However, we found susceptible children among younger ages and children with fewer or unknown IPV doses belonging to certain socioeconomic strata. Results are relevant for countries transitioning from OPV to IPV and underline the importance of achieving high coverage with IPV.

Analysis of an Imported Subgenotype C2 Strain of Human Enterovirus 71 in Beijing, China, 2015.

Background: Subgenotype C4 of enterovirus 71 (EV71) is the predominant agent of Hand Foot and Mouth disease (HFMD) circulating in the mainland of China. For the first time, a subgenotype C2 of EV71 named SY30-2 was isolated from a HFMD case in Beijing, China. Since it is uncertain whether antibodies raised against subgenotype C4 of EV71 can protect C2 EV71, it is important to monitor and check the presence of cross-reactive antibodies against new EV71 subgenotypes. To find out the causes for the different NtAb, this study is to investigate the relationships between amino acid residue variations and cross-reactive antibodies against EV71 subgenotypes C2 and C4.Methods: Nucleotide and amino acid sequences from full-length genome sequence of SY30-2 were compared to EV71 reference strains. A microneutralization test was used to detect neutralizing antibody (NTAb) in the sera of subgenotype C4 of EV71 infected cases against SY30-2 and FY17 (a C4 isolate). The 3D structure of the viral capsid protein of SY30-2 was constructed.Results: Genome sequence and similarity plot analyses showed that SY30-2 shared the highest identity with subgenotype C2 of EV71 strains in every fragment of the genome. While the microneutralization test result showed that children infected with subgenotype C4 of EV71 had higher NTAb titers against FY17 than SY30-2 (p < 0.001). The amino acid sequence comparison revealed that four amino acid residues VP1-22, VP1-31, VP1-249 and VP3-93 were highly conserved in subgenotype C4 of EV71 compared with the corresponding amino acid residues on subgenotype C2 of EV71 (p < 0.05). Furthermore, the 3D-structure of viral capsid protein showed that VP1-22, VP1-31 and VP3-93 were located on the surface of virion.Conclusion: This is the first report of an EV71 subgenotype C2 isolated from HFMD in Beijing, China. Only a few antigenic variations on subgenotype C2 of EV71 could have led to a great decrease in NTAb titer. Thus, imported new genotypes and subgenotypes of EV71 should be closely monitored. The efficacy of available vaccines against new viruses should be evaluated as well.

Cross-Protective Efficacy of Monovalent Live Influenza B Vaccines against Genetically Different Lineages of B/Victoria and B/Yamagata in Ferrets.

Background Currently, two genetic lineages of influenza B virus, B/Victoria and B/Yamagata, are cocirculating in humans in various countries. This situation has raised a question regarding the possibility of cross-protection between B components of live attenuated influenza vaccine (LAIV) belonging to different lineages. This study aimed to assess in naïve ferrets the potential protective activity of monovalent B-LAIVs against challenge with homologous and heterologous wild-type (WT) influenza B viruses. Methods Groups of seronegative female ferrets 5-6 months of age were given one dose of monovalent LAIV based on B/Victoria or B/Yamagata lineage virus. Ferrets were challenged 21 days later with B/Victoria or B/Yamagata WT virus. Ferrets were monitored closely for clinical signs and morbidity outcomes including febrile response, body weight loss, nasal symptoms, and level of activity one week prior to vaccination and for three days following vaccination/challenge. Nasal washes were collected three days after vaccination/challenge. Samples of lung tissue were taken three days after challenge. All samples were analyzed for the presence of challenge virus by culturing in embryonated chicken eggs and real-time polymerase chain reaction. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay and microneutralization test. Results Vaccination led to intensive production of specific neutralizing and antihemagglutinating antibodies to vaccine virus, protected ferrets from homologous challenge infection, and significantly reduced clinical signs and replication of homologous challenge virus. In contrast, cross-lineage serum antibodies were not detected. However, ferrets vaccinated with monovalent B-LAIV had a significantly lower level of heterologous challenge virus in the respiratory tract than those given challenge virus only. Conclusions Monovalent B-LAIV has the potential to be cross-protective against infection with genetically different influenza lineages. Further studies are required to confirm this effect.

Construction of human adenovirus type 4 vector expressing enhanced green fluorescence protein

Objective To prepare human adenovirus type 4 (Ad4) vector expressing enhanced green fluorescence protein (EGFP). Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombinant plasmid was transfected into AD293 cells to rescue recombinant virus which was identified by sequencing, SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP expressing EGFP was obtained and could be recognized and neutralized by monoclonal antibody MN4b and antisera against Ad4. The Ad4-specific and EGFP-specific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development, drug evaluation and transgene vector. Key words: Human adenovirus type 4; Vector; Enhanced green fluorescence protein; Neutralization

Assessment of Baseline Antibodies to Pandemic Influenza A/H1N1/2009 Virus in Ogbomoso, Oyo State, Nigeria

Acute respiratory infection remains a global leading cause of death, with influenza accounting one of the most important causes of severe infections and deaths every year [1]. Globally, an estimated 1 million persons died from respiratory and cardiovascular conditions associated with influenza A/H1N1/2009 infections. Transmission patterns of influenza are attached with unique epidemiologic and demographic features such as environmental, economic, religious, cultural and social conditions [2]; weather conditions such as temperate and humidity; pre-existing immunity accounting for difference in the prevalence rates of specific antibodies [3]. Serum antibody to influenza can be used to identify past exposure and measure current immune status. Various laboratory methods applied to determining the host’s immune response include haemagglutination inhibition (HI) test, neutralization test (NT), microneutralization (MN) test, and enzyme-linked immunosorbent assay (ELISA) [4]. Although both HI and NT tests serve as measures of antibody concentration in sera, they have important differences in how they are conducted and how they measure immunity. Haemagglutination inhibition and NT assays, despite their widespread usage, the difference in reliability between laboratories is a direct result of how they are been measured [5]. These assays assess the level of functional immunity to a virus in a similar manner, both using serial dilution of sera applied to a fixed amount of virus to determine at which titer of sera the virus is effectively inhibited [6], however, the difference is in the biological mechanism used as an indicator for inhibition [7]. The HI assay utilizes the natural process of viral hemagglutination, a process in which a lattice forms by binding of viruses to red blood cells; this process is blocked when sufficient antibody with affinity to the virus is present [8]. The NT assay, in contrast, measures cytopathic effects of the virus, the invading and killing of cells, through plaque formation [9]. Again, the antibodies in the sample serum are tested for their ability to block this activity. Results are expressed as reciprocal of the highest dilution at which virus infection is blocked. A more precise assessment of the total number of infected persons would require the large-scale use of techniques to specifically determine the presence and relative concentration of A/H1N1/2009 influenza virus antibodies in serum samples. Besides its obvious epidemiological significance, the availability of these techniques would also allow the rapid discernment of potentially immune subjects among those more susceptible to infection in a given population [5]. However, as a result of the somewhat time, labour intensive, specialized training, less availability of specific reagents needed for these techniques, hence, the assessment of specific baseline antibodies to A/H1N1/2009 influenza virus with an ELISA-based method for sero-epidemiological study among randomly-selected apparently healthy individuals. The immunoassay used for this study shows significant advantages over conventional MN and HI assays; (i) it does not require fresh chicken erythrocytes; (ii) it does not require virus manipulation and therefore does not require special infrastructure; and (iii) interpretation of results rests on absorbance readings, instead of subjective visual estimation of agglutination.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface.