Fluorescent Focus Units Research Articles

A New Gnotobiotic Pig Model of P[6] Human Rotavirus Infection and Disease for Preclinical Evaluation of Rotavirus Vaccines.

Human rotavirus (HRV) is a leading cause of gastroenteritis in children under 5 years of age. Licensed vaccines containing G1P[8] and G1-4P[8] strains are less efficacious against newly emerging P[6] strains, indicating an urgent need for better cross protective vaccines. Here, we report our development of a new gnotobiotic (Gn) pig model of P[6] HRV infection and disease as a tool for evaluating potential vaccine candidates. The Arg HRV (G4P[6]) strain was derived from a diarrheic human infant stool sample and determined to be free of other viruses by metagenomic sequencing. Neonatal Gn pigs were orally inoculated with the stool suspension containing 5.6 × 105 fluorescent focus units (FFU) of the virus. Small and large intestinal contents were collected at post inoculation day 2 or 3. The virus was passaged 6 times in neonatal Gn pigs to generate a large inoculum pool. Next, 33-34 day old Gn pigs were orally inoculated with 10-2, 103, 104, and 105 FFU of Arg HRV to determine the optimal challenge dose. All pigs developed clinical signs of infection, regardless of the inoculum dose. The optimal challenge dose was determined to be 105 FFU. This new Gn pig model is ready to be used to assess the protective efficacy of candidate monovalent and multivalent vaccines against P[6] HRV.

Defining the root cause of reduced H1N1 live attenuated influenza vaccine effectiveness: low viral fitness leads to inter-strain competition

In the 2013–14 and 2015–16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the H1N1 component of the FluMist quadrivalent live attenuated influenza vaccine (QLAIV) in the USA, leading to loss of Advisory Committee on Immunization Practices recommendation. Here we demonstrate in ferrets that 2015–16A/H1N1pdm09 vaccine strain A/Bolivia/559/2013 (A/BOL13) is outcompeted in trivalent (TLAIV) and QLAIV formulations, leading to reduced protection from wild-type challenge. While monovalent (MLAIV) A/BOL13 provided significant protection from wild-type virus shedding and fever at doses as low as 3.0 log10 fluorescent focus units (FFU), it failed to provide a similar level of protection in TLAIV or QLAIV formulation, even at a 6.0 log10 FFU dose. Conversely, clinically effective H1N1 strain A/New Caledonia/20/1999 provided significant protection in MLAIV, TLAIV, and QLAIV formulations. In conclusion, reduced A/BOL13 replicative fitness rendered it susceptible to inter-strain competition in QLAIV, contributing to its reduced VE in the 2015–16 season.

Evaluation of the Safety and Immunogenicity of a Candidate Pandemic Live Attenuated Influenza Vaccine (pLAIV) Against Influenza A(H7N9).

Background. We evaluated a candidate A/Anhui/2013(H7N9) pandemic live attenuated influenza vaccine (pLAIV) in healthy adults, and assessed the ability of 1 or 2 doses to induce immune memory. Methods. Healthy subjects in 2 age groups (18–49 years and 50–70 years) with undetectable hemagglutination-inhibiting (HAI) antibody to H7N9 were enrolled. Younger subjects received either 1 or 2 intranasal doses of 107.0 fluorescent focus units of A/Anhui/1/2013 pLAIV, while older subjects received a single dose. All subjects received a single 30-µg dose of unadjuvanted, antigenically matched A/Shanghai2/2013(H7N9) pandemic inactivated influenza vaccine (pIIV) 12 weeks after their first dose of pLAIV. Results. Both vaccines were well tolerated. Serum HAI antibody responses were detected in 0 of 32 younger subjects and 1 of 17 older subjects after 1 dose of pLAIV and in 2 of 16 younger subjects after a second dose. Strong serum antibody responses were detected after a single subsequent dose of pIIV that was broadly reactive against H7 influenza viruses. Conclusions. An A(H7N9) pLAIV candidate was safe in both age groups. Priming with pLAIV resulted in responses to subsequent pIIV that exceeded those seen in naive subjects in previous reports. The A(H7N9) pLAIV induces strong immune memory that can be demonstrated by exposure to subsequent antigenic challenge. Clinical Trials Registration. NCT01995695 and NCT02274545.

A comparative retrospective study of RT-PCR-based liquid hybridization assay for early, definitive diagnosis of dengue

Dengue is an important flaviviral infection in tropical and subtropical regions. Early diagnosis of dengue infection helps in monitoring the disease, determining when hospital admission is necessary and reducing case fatalities. The objective of this study was to carry out a retrospective comparison of an RT-PCR-based liquid hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of non-structural 3 gene were hybridized with a mixture of four dengue type-specific DNA probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 119 fever patients with or without dengue, confirmed by haemagglutination inhibition (HAI) assay, the gold standard assay for diagnosis of dengue infection. The RT-PCR-LH assay was highly specific for dengue and, as an early laboratory diagnostic method, had 100% sensitivity in detecting dengue patients confirmed by HAI assay. A high analytical sensitivity of two fluorescent focus units of dengue virus/reaction was achieved. This RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection when serological methods are of little value.

Concomitant administration of seasonal trivalent and pandemic monovalent H1N1 live attenuated influenza vaccines

To the editor: In the United States, live attenuated influenza vaccine (LAIV) will play a prominent role in the novel A(H1N1) pandemic vaccination campaign; more than 40 million doses have been purchased by the US Government. Additionally, approximately 10 million doses of seasonal trivalent LAIV will be available for use during the 2009–2010 influenza season. Seasonal trivalent LAIV is currently approved for use in eligible individuals aged 2–49 years in the United States, South Korea and Hong Kong. Because some US populations have been recommended to receive both seasonal trivalent and pandemic monovalent vaccine during the 2009–2010 influenza season, questions have arisen regarding concomitant vaccination with seasonal trivalent and pandemic monovalent LAIV. Historically, recommendations of the Centers for Disease Control and Prevention Advisory Committee on Immunization Practices (ACIP) have stated, ‘In the absence of specific data indicating interference, following ACIP’s general recommendations for vaccination is prudent. Inactivated vaccines do not interfere with the immune response to other inactivated vaccines or to live vaccines. Inactivated or live vaccines can be administered simultaneously with LAIV. However, after administration of a live vaccine, at least 4 weeks should pass before another live vaccine is administered’. 1 Specific guidance was published in 2009 related to vaccination with pandemic monovalent vaccines, which stated, ‘Simultaneous administration of inactivated vaccines against seasonal and novel influenza A (H1N1) viruses is permissible if different anatomic sites are used. However, simultaneous administration of LAIV against seasonal and novel influenza A (H1N1) virus is not recommended’. 2 It was subsequently noted that the recommendation against simultaneous intranasal administration of seasonal and pandemic LAIV was because of theoretical concerns for potential interference between the vaccines. 3 Here, we report the results of a study designed to examine the potential for interference following concomitant administration of seasonal and pandemic LAIV in ferrets, a widely accepted and relevant animal model often used to examine influenza virus and influenza vaccine immunogenicity, including annual World Health Organization and US Food and Drug Administration evaluation of candidate vaccine strains. 4 , 5 , 6 , 7 , 8 Twenty 8‐week‐old male ferrets (Triple F Farms, Sayre, PA, USA) seronegative for all four influenza strains were used in the study. One cohort (n = 4) was inoculated intranasally with a 0·2‐ml dose (0·1 ml per nostril) of seasonal trivalent LAIV containing 106·5−7·5 fluorescent focus units (FFU) of each of the three cold‐adapted, temperature‐sensitive vaccine strains recommended for inclusion in the 2009–2010 vaccine: A/South Dakota/6/2007 (H1N1) (A/Brisbane/59/2007‐like), A/Uruguay/716/2007 (H3N2) (A/Brisbane/10/2007‐like) and B/Brisbane/60/2008. A second cohort (n = 4) was inoculated intranasally with 106·5−7·5 FFU per dose of the cold‐adapted, temperature‐sensitive 2009 H1N1 monovalent vaccine, A/California/07/2009 (H1N1). A third group (n = 12) was inoculated intranasally with pandemic monovalent LAIV followed by seasonal trivalent LAIV approximately 15 seconds later. This third cohort included more animals to allow for further division into three subgroups to investigate second‐dose responses if interference was observed. Sera were collected weekly, and the immunogenicity and kinetics of the immune response were determined by strain‐specific serum hemagglutination inhibition (HAI) on days 0 (pre‐dose), 14 and 28 post‐inoculation using standard methods with 0·5% chicken erythrocytes. Cold‐adapted virus antigen was used for A/California/07/2009; wild‐type antigen was used for seasonal strains. Serum antibody responses to the four vaccine strains are depicted in Figure 1. All strains were immunogenic and strain‐specific responses were statistically similar in the cohorts receiving seasonal vaccine, pandemic vaccine and both vaccines concomitantly. No interference with concomitant vaccination was observed at either 14 or 28 days post‐vaccination. LAIV viruses replicate primarily in the ciliated epithelial cells of the nasopharyngeal mucosa to induce immune responses via mucosal immunoglobulin A (IgA), serum IgG and cellular immunity. Serum antibody responses are not a correlate of protection; in fact, some studies have shown protection in the absence of significant antibody responses. 9 , 10 , 11 However, consistent with their use in the present study, HAI responses have been used to establish comparability of different LAIV formulations and evaluate concomitant vaccination regimens. 12 , 13 , 14 Figure 1 Mean (log2) hemagglutination inhibition (HAI) titer for each vaccine strain 14 (A) and 28 days (B) after one dose of seasonal trivalent or pandemic monovalent H1N1 influenza vaccine or one dose of each vaccine administered concurrently. ... These data in ferrets indicate the development of a robust and consistent immune response at 14 and 28 days post‐inoculation with seasonal trivalent and pandemic monovalent H1N1 vaccines. There was no evidence of interference in the cohort receiving concomitant seasonal and pandemic vaccination. These data may help inform vaccination recommendations for the 2009–2010 influenza season in the United States. It should be noted that no data regarding concomitant vaccination exist for humans at this time.

Quantitation of flaviviruses by fluorescent focus assay

An indirect immunofluorescence assay for quantitation of flaviviruses was developed as an alternative to the standard plaque assay. The assay was validated with West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Dengue virus (DENV) types 1–4. Vero cells were plated in 8-well chamber slides, and infected with 10-fold serial dilutions of virus. About 1–3 days after infection, cells were fixed, incubated with specific monoclonal antibody, and stained with a secondary antibody labeled with a fluorescent tag. Fluorescent foci of infection were observed and counted using a fluorescence microscope, and viral titers were calculated as fluorescent focus units (FFU) per ml. The optimal time for performing the fluorescent focus assay (FFA) on Vero cells was 24 h for WNV, and 48 h for SLEV and the four DENV serotypes. In contrast, the time required to complete a standard Vero cell plaque assay for these viruses range from 3 days for WNV to 11 days for DENV-1. Thus, the FFA method of virus titration is useful for viruses whose plaques develop slowly. In addition, these viruses can be quantitated by FFA on a mosquito cell line (C6/36), which does not support plaque formation. The FFA for flaviviruses was validated for accuracy, precision, specificity, and robustness of the assay.

Detection of hepatitis A virus (HAV) in oysters ( Crassostrea gigas)

Because shellfish (oysters, clams, and mussels) are filter-feeders, pathogens become concentrated within them, and human consumption of raw, or under-cooked shellfish can result in disease outbreaks. Identification of hepatitis A virus (HAV) in shellfish has been difficult for several reasons: the concentration of virions in shellfish tissues are very low, detection methods based on in vitro propagation are unreliable, recovery of virions from shellfish tissues is inefficient, and PCR inhibitors in shellfish tissues limit the success of RT-PCR. These facts underlie difficulties in determining cause and effect relationships between hepatitis A outbreaks and detection of HAV contamination in shellfish samples. We have developed a reliable and highly sensitive method for detection of HAV in oyster tissues at low levels (0.001 FFU/ml—fluorescent focus units per milliliter). Our method combines dissection of the gastrointestinal oyster tract, organic extraction before PEG precipitation, and RNA extraction with Trizol LS ®, followed by RT-PCR and hybridization using a digoxigenin-labeled HAV cDNA probe. Our results will benefit both public health officials concerned about hepatitis A infections caused by consumption of HAV-contaminated oysters and shellfish producers who require reliable methods for quality control of commercial oyster production.

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l −1. We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l −1 and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 ± 0.2 × 10 6 cells ml −1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 ± 1.6 × 10 7 Fluorescent Focus Units (FFU) ml −1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 ± 0.5 × 10 6 cells ml −1. Cell infection under the conditions previously indicated, yielded 14 l of virus harvest that had a virus titer equal to 2.6 ± 0.5 × 10 7 FFU ml −1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.

Comparison of various culture modes for the production of rabies virus by Vero cells grown on microcarriers in a 2-l bioreactor

We studied Vero cell growth on Cytodex 1 (3 g/l) and MEM supplemented with foetal calf serum (FCS) in a 2-l bioreactor using continuous (recirculation and perfusion) and batch culture modes. We achieved a cell density level equal to 4.35 × 10 6 cells/ml using the recirculation culture mode while perfused cultures yielded a higher cell density level equal to 4.73 × 10 6 cells/ml. However, as compared to the recirculation culture mode, the specific medium consumption rate was two-fold higher. Batch culture of Vero cells resulted in 2.2 × 10 6 cells/ml with a specific medium consumption rate similar to recirculation culture. Rabies virus production (LP 2061/Vero strain) by Vero cells was also investigated. We analysed in spinner flasks, the effects on virus multiplication of multiplicity of infection (MOI), regulation of glucose level to 1 g/l and type of medium. The highest virus titer was reached when cells were infected at an MOI of 0.3 in M199 medium supplemented with 0.2% of bovine serum albumin (BSA) and without regulating glucose level. In these conditions, virus titer was equal to 1.5 × 10 6 fluorescent focus units (FFU)/ml. We then evaluated rabies virus production by Vero cells grown on 3 g/l of Cytodex 1 using recirculation culture mode during the growth phase. Cells were infected at the optimal conditions previously determined. The maximal virus titer was equal to 2 × 10 7 FFU/ml. The activity of the experimental vaccine prepared showed a protective activity that meets WHO requirements.

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor.

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.

Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pig model of human rotavirus disease.

Antibody responses to the Wa human rotavirus (HRV) nonstructural protein NSP4, a viral enterotoxin, were evaluated in neonatal gnotobiotic (Gn) pigs. Gn pigs were inoculated orally with one dose of 10(5) fluorescent focus units (FFU) of virulent Wa HRV (HRV-V), to mimic natural infection, or with three doses of 5 x 10(7) FFU attenuated Wa HRV (HRV-A) at 10-day intervals, to mimic oral attenuated rotavirus vaccines, or they were mock inoculated (mock). Subsets of pigs were challenged with 10(6) FFU of virulent Wa HRV at post-inoculation day 28 (PID 28). Post-challenge, the HRV-V pigs were completely protected against diarrhea and virus shedding, whereas the HRV-A pigs had a 50% protection rate against diarrhea and a 67% protection rate against virus shedding. All mock-inoculated pigs shed virus and had diarrhea post-challenge. Isotype antibody titers to NSP4 were compared in serum and intestinal contents, at post-inoculation day (PID) 28 and at post-challenge day 7 (PCD 7/PID 35) by indirect ELISA, using purified recombinant NH2-6xHis-tagged NSP4 of virulent Wa HRV. Pre-challenge, both the HRV-V and HRV-A-inoculated pigs had similar moderate titers of serum IgG antibodies to NSP4. However, only the HRV-V-inoculated pigs developed detectable serum and intestinal IgA antibody titers to NSP4 pre-challenge, compared with the HRV-A-inoculated pigs. The mock-inoculated pigs had no IgM, IgA, or IgG antibodies to NSP4 pre-challenge. All Wa HRV-inoculated pigs developed low to moderate titers of serum IgM, IgG, and IgA antibodies to NSP4 post-challenge, but the mock-inoculated pigs had only IgM antibodies post-challenge. Both Wa HRV-inoculated groups developed low titers of IgA antibody to NSP4 in the small intestinal contents post-challenge, but titers were 5.8-fold higher in the HRV-V pigs. Our results concur with findings that both rotavirus vaccinated and naturally infected children seroconvert with modest IgG antibodies to NSP4 [Johansen et al. (1999) J Med Virol 59:369-367]. These data suggest that Gn pigs could be a useful model to evaluate serum and intestinal IgA antibodies to NSP4 and their role in protection against HRV infection. Further experiments may clarify whether (1) the NSP4 antibodies detected pre-challenge in the HRV-V pigs contribute to the higher protection rates observed, or (2) the reduced or delayed NSP4 antibody responses of the HRV-A pigs are associated with the lower protection rates in these pigs.

Microplate-reverse hybridization method to determine dengue virus serotype

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3′-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Aluminosilicates Enhance the Infectivity of Cytomegalovirus in Urine Using Centrifugation-enhanced Antigen Detection Technology

Due to the inherent lability of CMV, necessary laboratory identification of this infectious agent is often compromised by a delay in specimen transport. Previous studies have addressed the phenomenon of infectivity enhancement/reduction in the rate of infectivity loss by the incorporation into various viral assay systems of trace concentrations of the adsorbents montmorillonite (bentonite [M]) or kaolinite (kaolin [K]). We extended these studies to the clinical setting to identify whether such aluminosilicates would effect an enhanced level of CMV infectivity. The shell vial assay-indirect immunofluorescent assay (SVA-IFA) was utilized in comparative testing throughout this study. The addition of trace concentrations of M or K to the SVA-IFA was found to enhance the infectivity of CMV in urine by 115 and 126%, respectively. The total CMV detection rate by SVA-IFA was 29% (30/105). Three of the 30 (10%) CMV positive specimens were detected only in shell vials which had been supplemented with K or M. Two specimens were isolation positive alone. The addition of K or M to shell vials immediately prior to the start of the SVA-IFA has the potential of (a), enhancing assay readability by increasing the number of fluorescent focus units per vial monolayer and (b), of detecting positive urine specimens with low viral titers which might otherwise not be identified using the conventional SVA-IFA procedure.

Antiviral susceptibility testing of cytomegalovirus from primary culture using shell vial assay to detect the late viral antigen

Susceptibility testing of 68 cytomegalovirus (CMV) peripheral blood isolates to Ganciclovir (DHPG) and 11 blood isolates to Foscarnet (PFA), was performed on primary culture isolates using the shell vial assay methodology (SVA-IFA, that is, quantitation of fluorescent focus units, FFUs), with an anti-CMV monoclonal antibody to the late viral antigen. A positive reaction in monolayer cultures of MRC-5 cells was characterized by cytoplasmic fluorescence with inclusions at both or more commonly off one end of the elongated fibroblast nucleus. Isolates from conventional MRC-5 tube cultures displaying a 1 + (10% cytopathic effect) were inoculated into shell vials containing DHPG concentrations of 0, 1.5, 3, 6, 12, or 24 μl/ml or shell vials containing 400, 500, 800, or 1200 μM PFA. The optimal readability of monolayers (expressed as FFUs per monolayer) occurred at 96 h after treatment with DHPG and at 36–48 h with PFA. Resistance to DHPG was determined at the concentration of antiviral agent necessary to reduce the number of FFUs to 90% or 50% of the control [that is, the 90% minimum inhibitiory concentration (MIC 90) or MIC 50]. Six of 68 isolates showed an MIC 90 > 12 or an MIC 50 > 1.5 μg/ml, and were considered DHPG resistant. Three of the six isolates were from AIDS patients with latestage disease who had never received DHPG therapy. All but one (specimen 2400) DHPG-resistant isolates revealed MIC 90 values to a PFA concentration of 500 μM, which is considered an achievable peak plasma level in patients undergoing PFA therapy. The single DHPG- and FPA-resistant isolate was obtained from a patient displaying marked clinical resistance to both drugs. The use of a monoclonal antibody to the CMV late antigen enables a pattern to be easily recognized by SVA-IFA, enabling anti-CMV susceptibility testing to be completed within days after primary isolation.