Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins.

Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically Differentiate the Infected from the Vaccinated Animals (DIVA). We developed an indirect ELISA using HeV-G expressed in Leishmania tarentolae and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from naïve, vaccinated and infected horses that either originated from a Hendra-virus free region, or had been pre-tested in validated diagnostic tests. Our data confirm the reliability of this approach, as HeV-N-specific antibodies were only detected in sera from infected horses, while HeV-G-specific antibodies were detected in infected and vaccinated horses with a high level of specificity and sensitivity. Given the excellent correlation of data obtained for German and Australian HeV-negative horses, we assume that this test can be applied for the testing of horse serum samples from a variety of geographical regions.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses.

Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics. At a 0.4 S/P cutoff, 16 of 19 sera from HeV-infected horses gave positive results in the HeV soluble G, indirect ELISA (HeVsG iELISA; DSe 84.2% [95% CI: 60.4-96.6%]); 463 of 477 non-infected horse sera tested negative (DSp 97.1% [95% CI: 95.1-98.4%]). The HeVsG iELISA eliminated almost all false-positive results from the previously used HeV iELISA, with marginally decreased relative sensitivity. Assay robustness was evaluated in inter-laboratory and proficiency testing panels. The HeVsG iELISA is considered to be fit for purpose for serosurveillance and international movement of horses when virus neutralization is used for follow-up testing of positive or inconclusive serum samples.

Salivary Blockade Protects the Lower Respiratory Tract of Mice from Lethal Influenza Virus Infection.

It is possible to model the progression of influenza virus from the upper respiratory tract to the lower respiratory tract in the mouse using viral inoculum delivered in a restricted manner to the nose. In this model, infection with the A/Udorn/307/72 (Udorn) strain of virus results ultimately in high viral titers in both the trachea and lungs. In contrast, the A/Puerto Rico/8/34 (PR8) strain causes an infection that is almost entirely limited to the nasal passages. The factors that govern the progression of virus down the respiratory tract are not well understood. Here, we show that, while PR8 virus grows to high titers in the nose, an inhibitor present in the saliva blocks further progression of infection to the trachea and lungs and renders an otherwise lethal dose of virus completely asymptomatic. In vitro, the salivary inhibitor was capable of potent neutralization of PR8 virus and an additional 20 strains of type A virus and two type B strains that were tested. The exceptions were Udorn virus and the closely related H3N2 strains A/Port Chalmers/1/73 and A/Victoria/3/75. Characterization of the salivary inhibitor showed it to be independent of sialic acid and other carbohydrates for its function. This and other biochemical properties, together with its virus strain specificity and in vivo function, indicate that the mouse salivary inhibitor is a previously undescribed innate inhibitory molecule that may have evolved to provide pulmonary protection of the species from fatal influenza virus infection.IMPORTANCE Influenza A virus occasionally jumps from aquatic birds, its natural host, into mammals to cause outbreaks of varying severity, including pandemics in humans. Despite the laboratory mouse being used as a model to study influenza virus pathogenesis, natural outbreaks of influenza have not been reported in the species. Here, we shed light on one mechanism that might allow mice to be protected from influenza in the wild. We show that virus deposited in the mouse upper respiratory tract will not progress to the lower respiratory tract due to the presence of a potent inhibitor of the virus in saliva. Containing inhibitor-sensitive virus to the upper respiratory tract renders an otherwise lethal infection subclinical. This knowledge sheds light on how natural inhibitors may have evolved to improve survival in this species.

Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B.

An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection.

Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.