Showing papers on "Viral culture published in 2017"

TLR3 Mutations in Adult Patients With Herpes Simplex Virus and Varicella-Zoster Virus Encephalitis

Abstract: Defects in genes of the Toll-like receptor 3 (TLR3) pathway are associated with susceptibility to herpes simplex virus type 1 encephalitis (HSE). We analyzed a cohort of 11 adult Italian patients in whom viral encephalitis developed. We detected 2 rare missense mutations in TLR3: 1 in a patient with HSE (p.Leu297Val) and 1 in a patient with varicella-zoster virus encephalitis (p.Leu199Phe). Both mutations are extremely rare in human populations and have pathogenicity scores highly suggestive of a functional effect. Data herein expand the phenotypic spectrum of TLR3 mutations to varicella-zoster virus encephalitis and support the role of TLR3 genetic defects as risk factors for HSE in adults.

Influenza in Children

Abstract: In children, influenza is one among the commonest causes of acute respiratory illness and loss of school days. Influenza A, B, and C are 3 types of viruses responsible for illness. Type A virus has many subtypes based on antigens but Type B and Type C viruses have no known subtypes. Currently, influenza A/H1N1, A/H3N2, and influenza type B viruses are circulating in humans. Transmission of influenza occurs through droplets from infected person or through direct contact with person or fomites. Clinically, influenza is characterized by acute onset fever, chills, running nose, cough, sore throat, headache and myalgia. Mostly, febrile illness lasts for 3-4 d with resolution of disease in 7-10 d. Confirmation of influenza can be done either by virus culture, RT-PCR or specific neutralizing antibodies in blood. Basic principles of management include prompt institution of infection control measures, early identification of children at higher risk, supportive care and antiviral drugs. Vaccine and chemoprophylaxis are two commonly used methods for prevention of influenza. Currently, inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV) are available for use with good efficacy. Cough etiquette, use of face masks and hand hygiene are the most important measures to reduce the risk of infection transmission from person to person.

Chikungunya Infection: a Global Public Health Menace

Abstract: Chikungunya virus (CHIKV) has been involved in epidemics in African and Asian subcontinents and, of late, has transcended to affect the Americas. Aedes aegypti and Aedes albopictus are the major vectors for CHIKV infection, which results in dissemination of virus to various vital organs. Entry of virus into these tissues causes infiltration of innate immune cells, monocytes, macrophages, neutrophils, natural killer cells, and adaptive immune cells. Macrophages bearing the replicating virus, in turn, secrete pro-inflammatory cytokines IL-1β, TNF-α, and IL-17. Together, this pro-inflammatory milieu induces osteoclastogenesis, bone loss, and erosion. CHIKV is characterized by fever, headache, myalgia, rash, and symmetric polyarthritis, which is generally self-limiting. In a subset of cases, however, musculoskeletal symptoms may persist for up to 3–5 years. Viral culture and isolation from blood cells of infected patients are the gold standards for diagnosis of CHIKV. In routine practice, however, assays for anti-CHIKV IgM antibodies are used for diagnosis, as elevated levels in blood of infected patients are noted from 10 days following infection for up to 3–6 months. Early diagnosis of CHIKV is possible by nucleic acid detection techniques. Treatment of acute CHIKV is mainly symptomatic, with analgesics, non-steroidal anti-inflammatory agents (NSAIDs), and low-dose steroids. No vaccines or anti-viral medicines have been approved for clinical therapy in CHIKV as yet. Hydroxychloroquine and methotrexate have been used in chronic CHIKV infection with variable success.

Label-Free Virus Capture and Release by a Microfluidic Device Integrated with Porous Silicon Nanowire Forest.

Abstract: Viral diseases are perpetual threats to human and animal health. Detection and characterization of viral pathogens require accurate, sensitive, and rapid diagnostic assays. For field and clinical samples, the sample preparation procedures limit the ultimate performance and utility of the overall virus diagnostic protocols. This study presents the development of a microfluidic device embedded with porous silicon nanowire (pSiNW) forest for label-free size-based point-of-care virus capture in a continuous curved flow design. The pSiNW forests with specific interwire spacing are synthesized in situ on both bottom and sidewalls of the microchannels in a batch process. With the enhancement effect of Dean flow, this study demonstrates that about 50% H5N2 avian influenza viruses are physically trapped without device clogging. A unique feature of the device is that captured viruses can be released by inducing self-degradation of the pSiNWs in physiological aqueous environment. About 60% of captured viruses can be released within 24 h for virus culture, subsequent molecular diagnosis, and other virus characterization and analyses. This device performs viable, unbiased, and label-free virus isolation and release. It has great potentials for virus discovery, virus isolation and culture, functional studies of virus pathogenicity, transmission, drug screening, and vaccine development.

Revisiting the concept of a cytopathic viral infection.

Abstract: The outcome of a viral infection with respect to cellular fate is a fundamental aspect of viral biology. Different virus families have vastly different interactions with their host cells, but they have long been broadly grouped as causing either persistent or cytopathic infections. Persistent infections are established through a variety of mechanisms, but all result in a long-term infection of the host cell. Other viruses cause a lytic (or cytopathic) infection that leads to cellular death shortly after viral infection. But what if cellular death was not the only outcome of a cytopathic viral infection? What if additional cell fates not only occurred but also were important for viral pathogenesis? The focus of this review will be on the evidence for cellular survival after cytopathic virus infections and the importance of these uncharacterized cell populations.

Adenovirus type 4 respiratory infections with a concurrent outbreak of coxsackievirus A21 among United States Army Basic Trainees, a retrospective viral etiology study using next-generation sequencing

Abstract: Human adenoviruses (HAdV), in particular types 4 and 7, frequently cause acute respiratory disease (ARD) during basic military training. HAdV4 and HAdV7 vaccines reduced the ARD risk in U.S. military. It is important to identify other respiratory pathogens and assess their potential impact on military readiness. In 2002, during a period when the HAdV vaccines were not available, throat swabs were taken from trainees (n = 184) with respiratory infections at Fort Jackson, South Carolina. Viral etiology was investigated initially with viral culture and neutralization assay and recently in this study by sequencing the viral isolates. Viral culture and neutralization assays identified 90 HAdV4 isolates and 27 additional cultures that showed viral cytopathic effects (CPE), including some with picornavirus-like CPE. Next-generation sequencing confirmed these results and determined viral genotypes, including 77 HAdV4, 4 HAdV3, 1 HAdV2, 17 coxsackievirus A21 (CAV21), and 1 enterovirus D68. Two samples were positive for both HAdV4 and CAV21. The identified genotypes are phylogenetically close to but distinct from those found during other years or in other military/non-military sites. HAdV4 is the predominant respiratory pathogen in unvaccinated military trainee. HAdV4 has temporal and demographic variability. CAV21 is a significant respiratory pathogen and needs to be evaluated for its current significance in military basic trainees.

A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease

Abstract: Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome replication-deficient Sendai vector that has many favorable vaccine characteristics. The specific vaccine design guarantees genetic stability of the transgene; furthermore, it supports a favorable presentation of the antigen, activating the adaptive response, features that other vectored vaccine approaches have often had difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies, and it could also be applied to other infectious diseases.

Simultaneous detection of respiratory syncytial virus and human metapneumovirus by one-step multiplex real-time RT-PCR in patients with respiratory symptoms.

Abstract: Both respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance. In order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay. Our one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously. In summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

Mild respiratory illness among young children caused by highly pathogenic avian influenza A (H5N1) virus infection in Dhaka, Bangladesh, 2011.

Abstract: Background In March 2011, a multidisciplinary team investigated 2 human cases of highly pathogenic avian influenza A(H5N1) virus infection, detected through population-based active surveillance for influenza in Bangladesh, to assess transmission and contain further spread. Methods We collected clinical and exposure history of the case patients and monitored persons coming within 1 m of a case patient during their infectious period. Nasopharyngeal wash specimens from case patients and contacts were tested with real-time reverse-transcription polymerase chain reaction, and virus culture and isolates were characterized. Serum samples were tested with microneutralization and hemagglutination inhibition assays. We tested poultry, wild bird, and environmental samples from case patient households and surrounding areas for influenza viruses. Results Two previously healthy case patients, aged 13 and 31 months, had influenzalike illness and fully recovered. They had contact with poultry 7 and 10 days before illness onset, respectively. None of their 57 contacts were subsequently ill. Clade 2.2.2.1 highly pathogenic avian influenza H5N1 viruses were isolated from the case patients and from chicken fecal samples collected at the live bird markets near the patients' dwellings. Conclusion Identification of H5N1 cases through population-based surveillance suggests possible additional undetected cases throughout Bangladesh and highlights the importance of surveillance for mild respiratory illness among populations frequently exposed to infected poultry.

Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

Abstract: For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

Enhanced production of enveloped viruses in BST‐2‐deficient cell lines

Abstract: Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc.

Management of Herpes in Pregnancy

Abstract: Herpes virus causes painful vesicles with HSV-1 primarily causing oral lesions and HSV-2 causing genital lesions on the vagina, vulva, or cervix. Diagnosis is made either by viral culture or nucleic acid detection. Primary outbreaks are associated with much higher rates of neonatal HSV infection than recurrent outbreaks. If there are any active lesions or prodromal symptoms present during labor, delivery should be performed by cesarean section [1].

Erratum to: A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

Abstract: Metagenomic approaches to detect viral genomes and variants in clinical samples have various challenges, including low viral titers and bacterial and human genome contamination. To address these limitations, we examined a next-generation sequencing (NGS) and iterative mapping approach for virus detection in clinical samples. We analyzed 40 clinical specimens from hospitalized children diagnosed with acute bronchiolitis, croup, or respiratory tract infections in which virus identification by viral culture or polymerase chain reaction (PCR) was unsuccessful. For our NGS data analysis pipeline, clinical samples were pooled into two NGS groups to reduce sequencing costs, and the depth and coverage of assembled contigs were effectively increased using an iterative mapping approach. PCR was individually performed for each specimen according to the NGS-predicted viral type. We successfully detected previously unidentified respiratory viruses in 26 of 40 specimens using our proposed NGS pipeline. Two dominant populations within the detected viruses were human rhinoviruses (HRVs; n = 14) and human coronavirus NL63 (n = 8), followed by human parainfluenza virus (HPIV), human parechovirus, influenza A virus, respiratory syncytial virus (RSV), and human metapneumovirus. This is the first study reporting the complete genome sequences of HRV-A101, HRV-C3, HPIV-4a, and RSV, as well as an analysis of their genetic variants, in Taiwan. These results demonstrate that this NGS pipeline allows to detect viruses which were not identified by routine diagnostic assays, directly from clinical samples.

Seroepidemiological and phylogenetic characterization of neurotropic enteroviruses in Ireland, 2005‐2014

Abstract: Enteroviruses (EVs) are associated with a broad spectrum of clinical presentation, including aseptic meningitis (AM), encephalitis, hand, foot and mouth disease, acute flaccid paralysis, and acute flaccid myelitis. Epidemics occur sporadically and are associated with increased cases of AM in children. The present study describes the seroepidemiological analysis of circulating EVs in Ireland from 2005 to 2014 and phylogenetic characterization of echovirus 30 (E-30), enterovirus A71 (EV-A71), and enterovirus D68 (EV-D68). EV VP1 genotyping was applied to viral isolates and clinical samples, including cerebrospinal fluid (CSF), and those isolates that remained untypeable by neutralising anti-sera. An increase in AM cases from 2010 to 2014 was associated with an E-30 genogroup variant VII and sequences clustered phylogenetically with those detected in AM outbreaks in France and Italy. EV-D68 viral RNA was not detected in CSF samples and no neurological involvement was reported. Three EV-A71 positive CSF samples were identified in patients presenting with AM. A phylogenetic analysis of respiratory-associated EV-D68 and EV-A71 cases in circulation was performed to determine baseline epidemiological data. EV-D68 segregated with clades B and B(1) and EV-A71 clustered as subgenogroup C2. The EV VP1 genotyping method was more sensitive than neutralising anti-sera methods by virus culture and importantly demonstrated concordance between EV genotypes in faecal and CSF samples which should facilitate EV screening by less invasive sampling approaches in AM presentations.

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus.

Abstract: Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.

Genetic Stability of Parainfluenza Virus 5-Vectored Human Respiratory Syncytial Virus Vaccine Candidates after In Vitro and In Vivo Passage.

Abstract: Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines.IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

Herpes simplex type 1 pneumonitis and acute respiratory distress syndrome in a patient with chronic lymphatic leukemia: a case report.

Abstract: Pulmonary pathogenicity of herpes simplex virus type 1 in patients in intensive care without classic immunosuppression as well as the necessity of antiviral treatment in the case of herpes simplex virus detection in respiratory specimens in these patients is controversial. We present a case of acute respiratory distress syndrome in a patient with stable chronic lymphatic leukemia not requiring treatment, in whom we diagnosed herpes simplex virus type 1 bronchopneumonitis based on herpes simplex virus type 1 detection in bronchoalveolar lavage fluid and clinical response to antiviral treatment. A 72-year-old white man presented with symptoms of lower respiratory tract infection. His medical history was significant for chronic lymphatic leukemia, which had been stable without treatment, arterial hypertension, multiple squamous cell carcinomas of the scalp, and alcohol overuse. Community-acquired pneumonia was suspected and appropriate broad-spectrum antibacterial treatment was initiated. Within a few hours, rapid respiratory deterioration led to cardiac arrest. He was successfully resuscitated, but developed acute respiratory distress syndrome. Furthermore, he remained febrile and inflammation markers remained elevated despite antibacterial treatment. Polymerase chain reaction from bronchoalveolar lavage fluid and viral culture from tracheobronchial secretions tested positive for herpes simplex virus type 1. We initiated antiviral treatment with acyclovir. Concomitantly we further escalated the antibacterial treatment, although no bacterial pathogen had been isolated at any point. Defervescence occurred rapidly and his C-reactive protein and leukocyte levels decreased. He was successfully weaned from mechanical ventilation, transferred to the ward, and eventually discharged to home. Herpes simplex virus should be considered a cause for lower respiratory tract infection in critically ill patients, especially in the setting of an underlying disease.

Exploring inhibitor structural features required to engage the 216-loop of human parainfluenza virus type-3 hemagglutinin-neuraminidase

Abstract: Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for inhibitor development. In this study, we explore the structural features required for Neu2en derivatives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neuraminidase protein.

Evaluation of viral etiology in central nervous system infections from a university hospital point of view in Izmir based on seven years data

Abstract: The serious diseases of the central nervous system (CNS); encephalitis and meningitis, have high mortality and morbidity rate especially not diagnosed and treated in time. Nucleic acid testing (NAT) is the tool of choice for viral diagnosis in CNS infections. In this study, viral etiological agents found in cerebrospinal fluid (CSF) samples sent to our university hospital virology laboratory for laboratory diagnosis of CNS infections were retrospectively evaluated and results were compared with other reports from our country. Viral etiological agents found in cerebrospinal fluid (CSF) samples sent to Ege University Faculty of Medicine Department of Medical Microbiology Virology Laboratories for laboratory diagnosis of CNS infection between 01.01.2009-31.12.2015 were evaluated retrospectively. A total of 3778 CSF tests were performed for cell culture of enterovirus (EV) in 487 samples and 3291 tests for nucleic acid testing (NAT) by real time polymerase chain reaction (PCR) in herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV6) and EV. VZV and EV NAT's were performed during the last one and five years period, respectively. NAT positive results for HSV1, HSV2, CMV, EBV, VZV, HHV6 and EV were 1.80% (24/1333), 0.08% (1/1333), 3.28% (19/580), 4.35% (22/506), 0.46% (1/216), 1.05% (5/478) and 3.37% (6/178), respectively. EV was isolated in 30 (6.20%) of 487 CSF samples by viral culture. Positive samples were mainly from pediatric, neurology and infectious diseases clinics as expected. The number of higher positive results were found in samples sentin december (35.3%), july (12.9%) and november (10.6%). Overall 80% of positive samples belonged to patients over 18 years old. When the results of other studies reported from Turkey are examined, although the positivity rates are generally similar, it is seen that the rates specific to certain factors are higher in selected smaller patient groups like HSV1 and EV. Rapid nucleic acid tests like multiplex PCR and microarray will provide more practical and effective laboratory diagnosis approach in CNS infections, since many more microorganisms may be causative agents.

Parainfluenza virus type 3 outbreak in a neonatal intensive care unit.

Abstract: Parainfluenza virus (PIV) is a respiratory pathogen in young children and is second only to the respiratory syncytial virus (RSV) as a cause of lower respiratory tract infection. PIV type 3 (PIV3) is the most severe. Herein we describe an outbreak of PIV3 in three infants in a neonatal intensive care unit. They were diagnosed on virus culture from pharyngeal swabs. We prevented the spread of the virus using standard infection control procedures and isolation of the symptomatic infants. One infant had severe chronic lung disease and was complicated with recurrent wheezing for a long time. Because RSV and PIV have many structural, pathogenic, epidemiologic, and clinical similarities, we speculate that PIV infection causes recurrent wheezing, as observed with RSV infection. Therefore, physicians must consider recurrent wheezing at the time of treatment of PIV infection early in life.

Comparison of Reverse Transcription-PCR and Viral Culture for detection of Respiratory Syncytial Virus in Young Children: Relation to Epidemiological and Clinical Findings

Abstract: Background: RSV is the most frequently identified agent responsible for LRTIs especially bronchiolitis worldwide. Multiple methods are used for the laboratory diagnosis of viral infections, including viral culture, antigen detection, nucleic acid detection, and serology. Morbidity and mortality of RSV infection are higher in infants suffering from concomitant disease as prematurity, congenital heart disease and others. Objectives: To compare the value of Reverse-Transcription Polymerase Chain Reaction (RT-PCR) and Conventional Viral Culture in diagnosis of RSV, to determine the incidence of RSV in pediatric respiratory illness and patients’ findings and to determine the mortality rate of RSV infection with concomitant disease. Methodology: This study was conducted on Ninety seven children (30 females and 67 males aged from 0 month up to 5 Years and were presenting with clinical pictures suggesting of LRTI (acute bronchiolitis or pneumonia). All patients were subjected to history taking, complete medical examination and thorough laboratory and radiological investigations. Tissue culture and RT-PCR were done on nasopharyngeal aspirates collected from all of these patients and the results of the two methods were compared with patient's demographic data, clinical and radiological findings to determine the best method for detection of RSV. Results: RSV was detected in 68.04% and 30.9% of LRTI cases by using RT-PCR and tissue culture respectively and there was a highly significant statistical difference (P value= 0.0001).The highest isolation rate of RSV (56.7% and 40.9%) was in children with age from 0-6m by tissue culture and RT-PCR respectively. The incidence of RSV was decreasing by increasing the age of the patients. By both techniques for RSV isolation, RSV was higher in males than females but there was no statistically significant difference. Sixty–six positive RSV cases detected by RT-PCR were distributed as the following in both seasons; 43.94% in January, 31.82%, in December, 22.73% in February and only 1.51%, in November. RSV was isolated from 92.7% of 61 cases of bronchiolitis by RT-PCR and the presence of hyperinflated chest is significantly higher among positive RSV cases (P value=0.001). Mortality rate in positive RSV patients with comorbidities was 25%. Conclusion: RT-PCR was more sensitive than tissue culture in diagnosis of RSV, especially in older age group cases. Mortality rate was higher among positive RSV who had concomitant diseases.

Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR.

Abstract: Background: Enterovirus (EV) and herpes simplex virus 1 and 2 (HSV1 and HSV2) are the main etiologic agents of central nervous system infections. Early laboratory confirmation of these infections is performed by viral culture of the cerebrospinal fluid (CSF), or the detection of specific antibodies in serum (e.g., HSV). The sensitivity of viral culture ranges from 65 to 75%, with a recovery time varying from 3 to 10 days. Serological tests are faster and easy to carry out, but they exhibit cross-reactivity between HSV1 and HSV2. Although molecular techniques are more sensitive (sensitivity >95%), they are more expensive and highly susceptible to cross-contamination. Methods: A real-time RT-PCR for the detection of EV, HSV1, and HSV2 was compared with end-point nested PCR. Results: We tested 87 CSF samples of patients with a clinical diagnosis of viral meningitis or encephalitis. Fourteen samples were found to be positive by RT-PCR, but only 8 were positive by end-point PCR. The RT-PCR showed a specificity range of 94-100%, the negative predictive value was 100%, and the positive predictive value was 62, 100, and 28% for HSV1, HSV2, and EV, respectively. Conclusion: Real-time RT-PCR detected EV, HSV1, and HSV2 with a higher sensitivity and specificity than end-point nested RT-PCR.

Method of producing porcine pseudorabies virus antigen by fully suspended cell culture

Abstract: The invention relates to a method of producing a porcine pseudorabies virus antigen by a fully suspended cell culture. The method comprises the following steps: 1) recovering a BHK21 cell, performing suspension culture amplification with a shake flask, and inoculating the BHK21 cell to a reactor for suspended culture with a low-serum culture medium to obtain a suspension culture cell sap; 2) replacing the low-serum culture medium in the suspension culture cell sap with a serum-free culture medium for further cultivation, and performing exposure for virus culture; 3) continuously cultivating the BHK21 suspended cell for 96-144h till dead cell quantity reaches 90% above, harvesting the cell suspended liquid, freeze thawing, centrifugalizing and collecting the supernatant to obtain a virus liquid; and 4) concentrating, deactivating and degerming the virus liquid to obtain the porcine pseudorabies virus antigen. A porcine pseudorabies virus antigen vaccine obtained by the method is high in titer which is stabilized at 108.5TCID50/mL above, and the porcine pseudorabies virus antigen vaccine is high in purity and stable in quality, and the product quality is remarkably improved.