Titers Of Infectious Virus Research Articles

The Intracellular Cargo Receptor ERGIC-53 Is Required for the Production of Infectious Arenavirus, Coronavirus, and Filovirus Particles

SummaryArenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

Comparison of the Mosquito Inoculation Technique and Quantitative Real Time Polymerase Chain Reaction to Measure Dengue Virus Concentration

An accurate measure of infectious dengue virus in human and mosquito tissues is critical to fully understand virus-host relationships, disease severity, viral fitness, and pathogenesis. In recent years, RNA copy number measured by quantitative real time-polymerase chain reaction has been used to measure dengue virus concentration in vitro and in vivo. In this study, we detail important differences in the measurement of viral growth kinetics in Vero and C6/36 tissue cultures, in Aedes aegypti mosquitoes, and in viremic human sera using RNA genomic equivalents and mosquito infectious dose 50 (MID50). Although there was reasonably good correlation between the two methods, RNA copy number was 2 to 5 logs greater than infectious virus titers. These differences varied significantly depending on virus strain, viral platform, infectious virus assay, and viral growth phase. The results have important implications for the correct interpretation of biological and epidemiological data from experimental and clinical studies, and show that genomic equivalents should be interpreted with caution when used as a proxy for infectious virus in such studies.

Rotavirus Replication in the Cholangiocyte Mediates the Temporal Dependence of Murine Biliary Atresia

Biliary atresia (BA) is a neonatal disease that results in obliteration of the biliary tree. The murine model of BA, which mirrors the human disease, is based upon infection of newborn mice with rhesus rotavirus (RRV), leading to an obstructive cholangiopathy. The purpose of this study was to characterize the temporal relationship between viral infection and the induction of this model. BALB/c mice were infected with RRV on day of life (DOL) 0, 3, 5, and 7. Groups were characterized as early-infection (infection by DOL 3) or late-infection (infection after DOL 5). Early RRV infection induced symptoms in 95% of pups with a mortality rate of 80%. In contrast, late infection caused symptoms in only 50% of mice, and 100% of pups survived. The clinical findings correlated with histological analysis of extrahepatic biliary trees, cytokine expression, and viral titers. Primary murine cholangiocytes isolated, cultured, and infected with RRV yielded higher titers of infectious virus in those harvested from DOL 2 versus DOL 9 mice. Less interferon alpha and beta was produced in DOL 2 versus DOL 9 RRV infected primary cholangiocytes. Injection of BALB/c interferon alpha/beta receptor knockout (IFN-αβR−/−) pups at DOL 7 showed increased symptoms (79%) and mortality (46%) when compared to late infected wild type mice. In conclusion, the degree of injury sustained by relatively immature cholangiocytes due to more robust RRV replication correlated with more severe clinical manifestations of cholangiopathy and higher mortality. Interferon alpha production by cholangiocytes appears to play a regulatory role. These findings confirm a temporal dependence of RRV infection in murine BA and begin to define a pathophysiologic role of the maturing cholangiocyte.

Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein

Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer.

Eradicating bovine viral diarrhoea virus

BOVINE viral diarrhoea virus (BVDV) causes widespread infection of cattle populations worldwide and, even though the majority of infections go unnoticed, is an important disease (Brownlie and others 2000). BVDV...

Sustained replication of dengue pseudoinfectious virus lacking the capsid gene by trans-complementation in capsid-producing mosquito cells

A simple system for the generation of pseudoinfectious particles of dengue virus was developed to facilitate studies of virus replication and vaccine development. Selected clones of the C6/36 mosquito cell line expressing an anchored form of the dengue virus capsid protein served as host cells for the trans-complementation of partially capsid-deleted viral RNA generated in vitro. Transfection of the partially capsid-deleted viral RNA into the anchored capsid-expressing C6/36 cells resulted in moderate titers of infectious virus. Progeny viruses multiplied in the capsid trans-complementing C6/36 cells for up to three weeks, but only initiated single rounds of replication in Vero cells lacking the capsid protein. Employing this trans-complementation system, it was found that nearly all of the capsid-coding sequence in the viral RNA was dispensable for the generation of pseudoinfectious dengue virus particles in mosquito cells.

Genetic and Functional Characterization of the N-Terminal Region of the Hepatitis C Virus NS2 Protein

The hepatitis C virus (HCV) NS2 protein has dual roles within the HCV life cycle. While well characterized as an autoprotease that cleaves the NS2/NS3 junction, NS2, primarily via its N-terminal region, is also involved in virion morphogenesis. In order to map the determinants necessary for infectious virus production and gain further insight into the multiple points at which NS2 may impact this process, a detailed mutational analysis of residues spanning amino acids (aa) 1 to 92 was performed. Initial block mutagenesis (5 or 7 amino acid residues) in both bicistronic and monocistronic HCV cell culture-based (HCVcc) genomes revealed that all but two blocks had various levels of impaired infectious virus production. None of these mutations affected RNA replication, indicating that the N-terminal region of NS2 is not required for NS2-3 processing and replicase assembly. Fine mapping identified 29 critical residues that, when mutated, yielded at least a 1 log decrease in infectious virus titers. These mutants were characterized further with respect to release of extracellular HCV RNA and core, intracellular infectivity, thermal stability of virus particles, and NS2 interactions. While the most severely debilitated mutants were impaired early in the assembly process, which is in agreement with previous reports, others targeted later steps of virus production, most notably egress. Thus, in addition to participating in early steps in virion assembly, this comprehensive mutagenesis study suggests yet another role for NS2 in later steps in virus production.

Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors

The development of JFH1 based intergenotypic recombinants that exploit the unique replication characteristics of JFH1 has made it possible to study infectious hepatitis C virus (HCV) encoding the structural genes of additional HCV genotypes. To facilitate the study of 1b structural proteins, we aimed to develop a robust 1b/2a chimera encoding a humanized Renilla luciferase reporter gene (1b/2a hRluc). The unadapted genome replicated efficiently but produced very low titers of infectious virus. Adaptation by continuous passage over a novel Huh-7 Lunet clone improved viral titers approximately 100-fold but caused an unexpected decline in luciferase activity, limiting the utility of the reporter-containing virus. Genotypic analysis revealed 17 adenosine to guanosine (A to G) nucleotide mutations in the luciferase gene and two potential adaptive mutations. To overcome the problems of low viral titers and editing of the luciferase gene during viral adaptation, six adaptive mutations previously identified in a non-reporter 1b/2a HCV genome were introduced into the 1b/2a hRluc genome. This resulted in the immediate production of high-titer viral stocks (approximately 1000-fold greater than the parental virus) that could efficiently infect naïve cells and generate robust luciferase signals. The improved sensitivity of the luciferase reporter also facilitated time of addition studies validating the utility of this system for characterizing the early steps of HCV infection. Thus, the development of the 1b/2a hRluc reporter virus described here provides a versatile tool for discovery of inhibitors targeting the early steps of the viral life cycle and genotype 1b structural genes.

Venezuelan Equine Encephalitis Virus nsP2 Protein Regulates Packaging of the Viral Genome into Infectious Virions

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein. The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

T-705 (Favipiravir) Induces Lethal Mutagenesis in Influenza A H1N1 Viruses In Vitro

Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P < 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.

Site‐Specific Modification of Adeno‐Associated Viruses via a Genetically Engineered Aldehyde Tag

As a consequence of their well-defined nanostructure and intrinsic bioactive functionality, virus-based nanoparticles have shown promise for mediating gene delivery. Adeno-associated virus (AAV) nanoparticles, which possess an excellent safety profile and therapeutic potential, hold potential for use in human gene therapy. However, because of their native tropisms, the applicability of AAV nanoparticles is often limited to restricted ranges of cells or tissues. Thus, retargeting AAV particles to the desired cell populations has continued to be a major research focus in many gene therapy applications. In this study, a general strategy is reported for nanoparticle targeting. This involves the site-specific modification of AAV type 2 (AAV2) by genetically incorporating a short peptide, in this case an aldehyde tag, in the viral capsid. Such a tag can be exploited for site-specific attachment of targeting molecules and allows for further introduction of targeting antibodies or ligands. It is shown that this modification neither affects the level of infectious viral titer nor intracellular trafficking properties. Furthermore, the site-specific conjugation of targeting antibodies could significantly enhance viral transduction to those target cells that have otherwise exhibited very low permissiveness to AAV2 infection. This method also allows the functional incorporation of RGD peptides onto AAV2 for enhanced delivery with implications for cancer gene therapy.

A Cell Culture Adapted HCV JFH1 Variant That Increases Viral Titers and Permits the Production of High Titer Infectious Chimeric Reporter Viruses

The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses.

Impaired Surfactant Production by Alveolar Epithelial Cells in a SCID-hu Lung Mouse Model of Congenital Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.

Efficacy of a levulinic acid plus sodium dodecyl sulfate-based sanitizer on inactivation of human norovirus surrogates.

Human noroviruses are the most common etiologic agent of foodborne illness in the United States. The inability to culture human noroviruses in the laboratory necessitates the use of surrogate viruses such as murine norovirus (MNV-1) and feline calicivirus (FCV) for inactivation studies. In this study, a novel sanitizer of organic acid (levulinic acid) plus the anionic detergent sodium dodecyl sulfate (SDS) was evaluated. Viruses were treated with levulinic acid (0.5 to 5%), SDS (0.05 to 2%), or combinations of levulinic acid plus SDS (1:10 solution of virus to sanitizer). MNV-1 inoculated onto stainless steel also was treated with a 5% levulinic acid plus 2% SDS liquid or foaming solution. Log reductions of viruses were determined with a plaque assay. Neither levulinic acid nor SDS alone were capable of inactivating MNV-1 or FCV, resulting in a ≤0.51-log reduction of the infectious virus titer. However, the combination of 0.5% levulinic acid plus 0.5% SDS inactivated both surrogates by 3 to 4.21 log PFU/ml after 1 min of exposure. Similarly, MNV-1 inoculated onto stainless steel was reduced by >1.50 log PFU/ml after 1 min and by >3.3 log PFU/ml after 5 min of exposure to a liquid or foaming solution of 5% levulinic acid plus 2% SDS. The presence of organic matter (up to 10%) in the virus inoculum did not significantly affect sanitizer efficacy. The fact that both of the active sanitizer ingredients are generally recognized as safe to use as food additives by the U.S. Food and Drug Administration further extends its potential in mitigating foodborne disease.

Hepatitis C Virus (HCV) Induces Formation of Stress Granules Whose Proteins Regulate HCV RNA Replication and Virus Assembly and Egress

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus

A faster semi-automated 96-well microtiter plate assay to determine viral infectivity titers, or viral focal units (vfu), of equine infectious anemia virus (EIAV) stocks is described. Optimization of the existing method modernizes a classic virological technique for viral titer determination by quantitating EIAV in experimentally infected cells via a cell-based ELISA. To allow for automation, multiple parameters of the current assay procedures were modified resulting in an assay that required only one quarter the original amount of virus and/or serum for infectivity or neutralization assays, respectively. Equivalent reductions in the required volumes of tissue culture, cell processing, and protein detection reagents were also achieved. Additionally, the new assay decreased the time required from start to finish from 10 days to 6 days (viral titer) or 7 days (viral neutralization), while increasing the number of samples that can be processed concurrently by transition to a 96-well microtiter plate format and by automated counting.

Relationship between mycotoxicosis and calcium during preproduction period in layers

This study was conducted to examine field cases of mycotoxicosis (A flatoxicosis and ochratoxicosis) and their effects on calcium during the preproduction period of 1SA brown pullets. Birds were 12-15 weeks of age. The feed was subjected to analysis for mycotoxin and blood samples were laboratory diagnosed for infectious bronchitis and infectious bursal viruses antibody titer and for determination calcium level. Clinical signs of affected birds were rubbery bone, ruffled feather, paleness and high mortality. Necropsy findings of sick birds were characterized by muscle dehydration, enlarged livers yellowished, kidney enlargement and urate deposition. Histopathological features of liver consisted of capsular thickening hepatocellular necrosis, subcapsular infiltration with inflammatory kidneys cells showed swelling of tubular cells, deposition of calcium between kidney tubules with infiltration of inflammatory cells. ELISA test revealed the presence of 800 ppb aflatoxin and 100 ppb ochratoxin. Normal titers of infectious bronchitis virus and infectious bursal diseases antibodies were recorded. Low blood calcium level of 8.2 mg/dl was registered in the tested blood samples.

Prophylactic and therapeutic combination effects of rimantadine and oseltamivir against influenza virus A (H3N2) infection in mice

The combined effect of rimantadine and oseltamivir in a prophylactic context (therapy beginning 4h pre-virus infection) and therapeutic context (therapy started at 24h post-viral inoculation) course on influenza H3N2 virus infection in mice was studied. In the prophylactic course 5 and 10mg/kg/day rimantadine with 0.2 and 0.4mg/kg/day (25:1 dose ratio) oseltamivir showed a protection index (PI) of 79.6% and 75%, respectively and a mean survival time (MST) of 13.1 and 12.9days. The individual effects of the same doses ranged from 0% to 33.3% PI and 8.2 to 10.3days MST, respectively. Lung virus titers were decreased 630-fold in the combination-treated groups as compared to monotherapy and placebo groups. The reduction of surface lung pathology in combination-treated groups demonstrated a protective effect for the combination of both antivirals. In the therapeutic course 5 and 10mg/kg rimantadine combined with 0.2 and 0.4mg/kg oseltamivir showed no beneficial effect. At higher dosage (0.8, 1.6, 3.2mg/kg oseltamivir and 20, 40, 80mg/kg rimantadine) preserving the 25:1 ratio, the resultant PI ranged from 57.6% to 80.5% and the MST was 12.8–13.4days. Used alone at the same doses the compounds’ protection varied between 10.7% and 71.8% PI, MST 9.8–12.8days (8.7days in PBS control). Compared to vehicle and individual treatment, a decrease in infectious viral titers of up to 1000-fold and other viral pneumonia parameters were also recorded. The therapeutic effect of the drugs’ optimal effective doses combinations was characterized as synergistic. Survival of animals was 81.2–100% and MST was extended by 5–7days compared to placebos. Monotherapy protection was from 9.1% to a maximum of 56.5%, MST being prolonged only by 1.3–4.2days compared to 7.5days in the PBS control group. Lung viral titers were decreased 1445-fold for the most efficacious combination groups and a significant reduction in lung parameters was observed. These data emphasize that prophylactic and therapeutic courses using a combination of oseltamivir and rimantadine have a significant protective effect in mice experimentally infected with drug-sensitive influenza virus A (H3N2).

Replication and infectivity of a novel genotype 1b hepatitis C virus clone

Hepatitis C virus infection is a major public health problem because of an estimated 170 million carriers worldwide. Genotype 1b is the major subtype of HCV in many countries and is resistant to interferon therapy. Study of the viral life cycle is important for understanding the mechanisms of interferon resistance of genotype 1b HCV strains. For such studies, genotype 1b HCV strains that can replicate and produce infectious virus particles in cultured cells are required. In the present study, we isolated HCV cDNA, which we named the NC1 strain, from a patient with acute severe hepatitis. Subgenomic replicon experiments revealed that several mutations enhanced the colony-formation efficiency of the NC1 replicon. The full-length NC1 genome with these adaptive mutations could replicate in cultured cells and produce infectious virus particles. The density gradient profile and morphology of the secreted virus particles were similar to those reported for the JFH-1 virus. Further introduction of a combination of mutations of the NS3 and NS5a regions into the NC1 mutants further enhanced secreted core protein levels and infectious virus titers in the culture medium of HCV-RNA-transfected cells. However, the virus infection efficiency was not sufficient for autonomous virus propagation in cultured cells. In conclusion, we established a novel cell culture-adapted genotype 1b HCV strain, termed NC1, which can produce infectious virus when the viral RNA is transfected into cells. This system provides an important opportunity for studying the life cycle of the genotype 1b HCV.

Detection and quantification of influenza C virus in pediatric respiratory specimens by real-time PCR and comparison with infectious viral counts

BackgroundThe epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone. ObjectiveTo evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection. Study designThe primers and probe for real-time PCR were designed to amplify the conserved region of the nucleoprotein gene based on the aligned sequences of nine isolates from 1967 to 2010. Respiratory specimens from children collected between January 2010 and August 2010 were examined for the presence of influenza C virus by cell culture and real-time PCR. Specimens that were positive for the virus using real-time PCR were further examined using an infectivity assay with embryonated hen's eggs. ResultsOf the 1203 specimens examined, 34 (2.8%) tested positive for the influenza C virus by cell culture and 51 (4.2%) tested positive by real-time PCR. The mean viral load and infectivity titer in specimens that tested positive using cell culture were 3.97×108copies/ml and 5.43×105EID50/ml, respectively, and those in specimens that were negative using cell culture were 2.18×106copies/ml and 3.67×102EID50/ml, respectively. In the clinical specimens with viral loads less than 105copies/ml, it was not possible to isolate the virus using embryonated hen's eggs. The copy number-to-EID50 ratio of the clinical specimens was much higher, ranging from 32 to 278,000, than those of culture fluid, ranging from 2.3 to 13.5. ConclusionThe real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection.