Development of a new enzyme immunoassay for improved detection of rotavirus in fecal specimens of vaccinated infants.

BACKGROUND: Group A rotavirus is the most common cause of acute diarrhea in young children worldwide. A simple and rapid enzyme immunoassay (EIA) has been commonly used to detect rotavirus infection and evaluate rotavirus vaccines. Currently licensed commercial EIA kits have low sensitivity. A more sensitive detection of rotavirus can improve rotavirus diagnostics and vaccine efficacy studies. OBJECTIVE: A biotin-avidin based sandwich EIA was developed and compared with commercial EIA kits for improved detection of viral shedding in fecal samples from infants who received human rotavirus vaccine Rotarix in Mexico. STUDY DESIGN: A monoclonal antibody (mAb: 1D4) specific to human rotavirus group antigen VP6 was prepared and used to develop a biotin-avidin based sandwich EIA. This EIA was employed to test 128 fecal samples from vaccinated infants, in comparison with two commercial EIA kits using RT-PCR as a reference. RESULTS: A new biotin-avidin based sandwich EIA showed specific reaction to group A rotaviruses, but not to other enteric viruses. This new EIA had a detection rate of 36.7% for rotavirus antigen shedding in fecal specimens, which was two times higher (16.4%, 18.0%) than those from two commercial EIA kits. CONCLUSION: The new EIA had specificity and higher sensitivity than commercial kits. This new EIA has the potential to detect rotavirus at lower concentration in clinical specimens and thus should be further evaluated as a more sensitive kit for use in diagnostics and vaccine efficacy and effectiveness studies.

Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection.

BACKGROUND: Laboratory correlates of influenza vaccine protection can best be identified by examining people who are infected despite vaccination. While the importance of antibody to viral hemagglutinin (HA) has long been recognized, the level of protection contributed independently by antibody to viral neuraminidase (NA) has not been determined. METHODS: Sera from a controlled trial of the efficacies of inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV) were tested by hemagglutination inhibition (HAI) assay, microneutralization (MN) assay, and a newly standardized lectin-based neuraminidase inhibition (NAI) assay. RESULTS: The NAI assay detected a vaccine response in 37% of IIV recipients, compared with 77% and 67% of participants in whom responses were detected by the HAI and MN assays, respectively. For LAIV recipients, the NAI, HAI, and MN assays detected responses in 6%, 21%, and 17%, respectively. In IIV recipients, as NAI assay titers rose, the frequency of infection fell, similar to patterns seen with HAI and MN assays. HAI and MN assay titers were highly correlated, but NAI assay titers exhibited less of a correlation. Analyses suggested an independent role for NAI antibody in protection, which was similar in the IIV, LAIV, and placebo groups. CONCLUSIONS: While NAI antibody is not produced to a large extent in response to current IIV, it appears to have an independent role in protection. As new influenza vaccines are developed, NA content should be considered. CLINICAL TRIALS REGISTRATION: NCT00538512.

IgM, IgG, and IgA antibody responses to influenza A(H1N1)pdm09 hemagglutinin in infected persons during the first wave of the 2009 pandemic in the United States.

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.

Expression and characterization of human group C rotavirus virus-like particles in insect cells.

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.

Stability of varicella-zoster virus open reading frame 63.

The stability of varicella-zoster virus (VZV) open reading frame (ORF) 63 was analyzed by sequential passage of a virus strain in cell culture. VZV was propagated in culture for 1,206 passages. ORF63 from six passages (18, 220, 516, 730, 1060, and 1,206) was selected and sequenced. Among the six passages, only passage 1,206 showed point mutations at three locations: 551, 618 and 661. In addition, western blot analysis with anti-ORF63 monoclonal antibodies showed no discernable difference in the size of the ORF63 gene product from passage 18 and that from passage 1,206. These results indicate the stability of VZV ORF63 gene in culture over 1,206 passages.

Rapid identification and authentication of closely related animal cell culture by polymerase chain reaction.

Animal cell lines are important resources for research and diagnostic applications. Cross-contamination and misidentification of cell lines, however, can cause major problems for research (for example, false results that come from contamination cells may mislead the science). Hence, it is imperative to routinely monitor cell lines for identity and authenticity. Here, we extend our previous work on identification and authentication of animal cell culture by polymerase chain reaction (PCR) amplification and DNA sequencing. A PCR-based method for rapid identification and authentication of closely related cell lines was described. In this method, two new primers were designed based on high homology in the aldolase gene family. Used together with our previous primers, the combinations of primers were able to differentiate closely related species, including human from monkey and mouse from rat. This PCR assay provides a rapid, simple, sensitive, and cost-effective method for authentication of closely related cell lines.

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing.

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.