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To help inform global guidelines on infant feeding, this systematic review synthesizes evidence related to the presence of the Ebola virus (EBOV) in breast milk and its potential risk of viral transmission to the infant when breastfeeding. We relied on a comprehensive search strategy to identify studies including women with suspected, probable, or confirmed EBOV infection, intending to breastfeed or give breast milk to an infant. Our search identified 10,454 records, and after deduplication and screening, we assessed 148 full texts. We included eight studies reporting on 10 breastfeeding mothers and their children (one mother with twins), who provided breast milk samples for assessment. EBOV was detected via RT‐PCR or viral culture in seven out of ten breast milk samples. Four out of the five‐breastfed infants with EBOV‐positive breast milk were found positive for EBOV infection, and all of these EBOV‐positive infants died. Since previous reports have detected EBOV in tears, saliva, sweat, and contaminated surfaces, with the current evidence, it is not possible to conclude with certainty that breast milk was the main route of EBOV transmission., Understanding maternal‐to‐child transmission routes is key to lowering, and ultimately preventing, the exposure of Ebola virus (EBOV) in pediatric populations. We undertook a systematic review of the available scientific literature to determine whether EBOV can be transmitted through breast milk and to describe the outcomes of the infants that ingested EBOV laboratory‐confirmed breast milk.

Rapid diagnosis of influenza is important for early treatment and institution of control measures. In developing tropical countries such as Malaysia, influenza occurs all year round, but molecular assays and conventional techniques (such as immunofluorescence and culture) for diagnosis are not widely available. Rapid influenza diagnostic tests (RIDTs) may be useful in this setting. A total of 552 fresh respiratory specimens were assessed from patients with respiratory symptoms at a teaching hospital in Kuala Lumpur, Malaysia from November 2017 to March 2018. Two digital immunoassays (DIAs), STANDARD F Influenza A/B Fluorescence Immunoassay (STANDARD F) and Sofia Influenza A + B Fluorescence Immunoassay (Sofia) and one conventional RIDT (immunochromatographic assay), SD Bioline Influenza Ag A/B/A(H1N1) Pandemic rapid test kit (SD Bioline) were evaluated in comparison with a WHO-recommended reverse transcription quantitative PCR (RT-qPCR). Of the 552 samples, influenza A virus was detected in 47 (8.5%) and influenza B virus in 7 (1.3%). The digital immunoassays STANDARD F and Sofia had significantly higher overall sensitivity rates (71.7% and 70.6%, respectively) than the conventional RIDT SD Bioline and immunofluorescence/viral culture (55.8% and 52.8%, respectively). Sensitivity rates were higher for influenza A than influenza B, and specificity rates were uniformly high, ranging from 98% to 100%. Digital readout RIDTs can be used in tropical settings with year-round influenza if PCR is unavailable.

Conjunctivitis is a frequent ocular disorder caused by human adenoviruses (HAdVs). Only a few of the 45 HAdV-D species are associated with epidemic keratoconjunctivitis, including HAdV-D8. Nosocomial outbreaks due to HAdV-D8 have been rarely described, because keratoconjunctivitis cases are clinically diagnosed and treated without having to characterize the causative agent. Moreover, molecular typing is tedious when using classical techniques. In this study, a hospital outbreak of conjunctivitis caused by HAdV-D8 was characterized using the recently developed whole-genome sequencing (WGS) method. Of the 363 patients attending the Ophthalmology Department between July 13 and August 13, 2018, 36 may have acquired intrahospital conjunctivitis. Also, 11 of 22 samples sent to the Virology section were selected for WGS analysis. The WGS results revealed that 10 out of 11 HAdV-D8 strains were closely related. The remaining strain (Case 28) was more similar to a strain from an outbreak in Germany obtained from a public sequence database. WGS results showed that outbreak HAdV-D8 strains had a minimum percentage of identity of 94.3%. WGS is useful in a clinical setting, because it avoids carrying out viral culture or specific polymerase chain reaction sequencing. The public availability of sequence reads makes it easier to compare clusters in circulation. In conclusion, WGS can play an important role in standard routines to describe viral outbreaks.

Objective To determine if chikungunya virus persists in synovial fluid after infection, potentially acting as a causative mechanism of persistent arthritis. Methods We conducted a cross-sectional study of 38 Colombian participants with clinical chikungunya virus infection during the 2014-2015 epidemic who reported chronic arthritis and 10 location-matched controls without chikungunya virus or arthritis. Prior chikungunya virus infection status was serologically confirmed, and the presence of synovial fluid chikungunya virus, viral RNA, and viral proteins was determined by viral culture, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and mass spectrometry, respectively. Biomarkers were assessed by multiplex analysis. Results Patients with serologically confirmed chikungunya arthritis (33 of 38 [87%]) were predominantly female (82%) and African Colombian (55%) or white Colombian (33%), with moderate disease activity (mean ± SD Disease Activity Score in 28 joints 4.52 ± 0.77) a median of 22 months after infection (interquartile range 21-23 months). Initial symptoms of chikungunya virus infection included joint pain (97%), swelling (97%), stiffness (91%), and fever (91%). The most commonly affected joints were the knees (87%), elbows (76%), wrists (75%), ankles (56%), fingers (56%), and toes (56%). Synovial fluid samples from all patients with chikungunya arthritis were negative for chikungunya virus on qRT-PCR, showed no viral proteins on mass spectrometry, and cultures were negative. Case and control plasma cytokine and chemokine concentrations did not differ significantly. Conclusion This is one of the largest observational studies involving analysis of the synovial fluid of chikungunya arthritis patients. Synovial fluid analysis revealed no detectable chikungunya virus. This finding suggests that chikungunya virus may cause arthritis through induction of potential host autoimmunity, suggesting a role for immunomodulating agents in the treatment of chikungunya arthritis, or that low-level viral persistence exists in synovial tissue only and is undetectable in synovial fluid.

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.

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.

Case reportA captive breeding colony of 9 greater bilbies (Macrotis lagotis) exhibited mild upper respiratory signs and sudden deaths with 100% mortality over a 2-week period. Histologically, acute necrotising and erosive epithelial lesions throughout the upper respiratory system and bronchi were associated with eosinophilic intranuclear inclusion bodies. Inclusions were also present in hepatocytes and adrenocortical cells, but were not always associated with necrosis. Transmission electron microscopy of lung sections revealed nucleocapsids forming arrays within some nuclei. A pan-herpesvirus PCR yielded a 440-bp product, with sequencing confirming homology with the alphaherpesviruses. Viral culture in a marsupial cell line resulted in cytopathic effect consistent with an alphaherpesvirus.

Aim Influenza causes a large burden of disease in children. Point-of-care testing (POCT) can rapidly diagnose influenza with the potential to reduce investigation and hospital admission rates, but information on its use in an Australian setting is limited. Methods Through a retrospective review of laboratory-confirmed influenza cases presenting at a paediatric emergency department (ED) in 2009, we evaluated children diagnosed by POCT versus standard testing (direct fluorescent antibody, polymerase chain reaction or viral culture) and assessed differences in investigations, admission requirements, length-of-stay (LOS) in ED/hospital and antibiotic/antiviral prescription. The rate of serious bacterial infection was examined. Results Compared with standard testing (n = 65), children diagnosed by positive POCT (n = 236) had a shorter median hospital LOS by 1 day (P = 0.006), increased antiviral prescription (odds ratio 3.31, P < 0.001) and a reduction in the time to influenza diagnosis (2.4 vs. 24.4 h, P < 0.001); however, a negative POCT result (n = 63) resulted in delayed diagnosis (44.0 h, P = 0.001). POCT did not decrease LOS in ED. Interpretation of reductions in admission and investigations with POCT may be limited by possible confounding. Approximately 4% of influenza patients had a serious bacterial infection; urinary tract infections were commonest (2.7%), but no cerebrospinal fluid cultures were positive. A single positive blood culture was seen among 332 immunocompetent influenza patients. Conclusions Influenza diagnosis by POCT was quicker and reduced LOS of hospitalised children, whereas negative results delayed diagnosis. Negative POCT should not alter usual investigations if influenza remains suspected. A controlled prospective study during the influenza season is needed to clarify the direct benefits of POCT.

Formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine was developed in the 1960s. However, this vaccine does not prevent infection in RSV-naive recipients and has the paradoxical effect of increasing the severity of RSV illness following natural infection, which has been a major obstacle to developing RSV vaccines. Several experimental animal models for determining the cause of the severe symptoms in FI-RSV recipients have been developed. In the present study, cotton rats immunized with FI-RSV were challenged with RSV and histopathological findings and recovery of infectious virus were studied. Copy numbers of mRNA of Th1 and Th2 cytokines were measured in lung tissues to gain better understanding of their immune responses. Infiltration of inflammatory cells and prominent interstitial pneumonitis were observed in the FI-RSV group, as was induction of mRNA of Th2 cytokines such as IL-4, IL-10, IL-13 and RANTES. Rats immunized with recombinant measles virus expressing the RSV F protein (MVAIK/RSV/F) and those treated with anti-RSV mAb (palivizumab) showed very mild interstitial pneumonitis. Amounts of mRNA of IL-1β, IFN-γ and IL-4 were higher in the MVAIK/RSV/F group. Administration of palivizumab before RSV challenge decreased the severity of interstitial pneumonitis in the FI-RSV group. FI-RSV induced skewed Th2 responses, resulting in severe inflammatory responses.

Live virus vaccines are a critical component of worldwide vaccination strategy for reducing disease burden but often require complex biological production processes that are sensitive to many different factors, both known and often unknown. Prior application of high-throughput process development (HTPD) approaches to these processes has been hampered by a complex design space, low-throughput analytics, and challenges inherent in biosafety level 2 containment and asepsis in laboratory automation. In 2013, we initiated a project with HighRes Biosolutions to design and install an integrated high-throughput screening platform to enable HTPD for biosafety level 2 upstream process development studies. The system incorporates the necessary tools for performing cell and virus culture studies in microplates, as well as advanced analytical capabilities necessary for assessment of cell phenotype, product quality, and antigen yield. To date, we have applied this system to screen optimal media formulations and viral production conditions in support of two viral vaccine programs, with phenotypic assays performed as an integrated part of the workflow. This case study illustrates the power of HTPD in addressing large-scale biological screening challenges by narrowing a vast design space and identifying parameter interactions in live virus production processes.

Introduction Influenza is a major cause of morbidity and mortality in elderly persons. Fever is included in all standard definitions of influenza-like illness (ILI), yet older patients may have diminished febrile response to infection. Therefore, we examined the utility of various thresholds to define fever for case definitions of influenza in persons ≥65 years of age. Methods Data from two prospective surveillance studies for respiratory viral infection in adults hospitalized with acute cardiopulmonary illnesses with or without fever were examined. The highest temperature reported prior to admission or measured during the first 24 h after admission was recorded. The diagnosis of influenza was made by a combination of viral culture, reverse-transcription polymerase chain reaction, antigen testing, and serology. Results A total of 2410 subjects (66% ≥65 years of age) were enrolled; 281 had influenza (261 influenza A, 19 influenza B, and one mixed influenza A and B). The commonly used definition of ILI (fever ≥37·8°C and cough) resulted in 57% sensitivity and 71% specificity in older adults. Receiver operating characteristic curves examining the various temperature thresholds combined with cough and/or sore throat showed the optimal balance between sensitivity and specificity to be 37·9°C (AUC 0·71) and 37·3°C (AUC 0·66), in younger and older persons, respectively. Conclusion Clinical decision rules using the presence of cough and fever may be helpful when screening for influenza or empiric antiviral treatment when rapid influenza testing is not available; however, lower fever thresholds may be considered for elderly subjects.

Molecular techniques have represented a major step forward in the diagnosis of viral respiratory infections. They are considered highly sensitive and specific compared to conventional techniques. In this study two nucleic acid amplification tests (NAATs) were compared to conventional methods (immunofluorescence and viral culture). The aim of this work was to discuss the clinical interpretation of the results obtained by NAATs on the basis of the two-decade experience of our group and the literature. Eighty nasopharyngeal aspirates were collected from children under six years attended for acute respiratory illness at the pediatric emergency room of a third level Hospital. Both NAATs tested (Seeplex(®) and Clart(®)) showed an overall higher performance regarding sensitivity (76% and 90%, respectively). Compared to Seeplex(®), the Clart(®) system tripled the number of multiple detections (8 by Seeplex(®) vs. 25 by Clart(®)). In some specimens both NAATs detected different viruses. Given these discrepancies and the fact that detection of viral nucleic acids is not necessarily related to the current clinical syndrome, the interpretation of molecular results may not always be so straightforward. The pros and cons of NAATs should always be taken into account when giving a result.

Introduction Pulmonary infiltration and pleural effusion caused by permeability syndrome are the hallmark of pulmonary manifestation of dengue cases. Objective and Methods We report a 95-year-old chronic obstructive pulmonary disease case having dengue shock syndrome. Chest X-ray examination revealed diffuse lung infiltration. However, bilateral pneumotoceles were unexpectedly found in computed tomography (CT) images. Dengue virus type 2 infection was confirmed by virus culture, serology and reverse transcriptase-polymerase chain reaction. Profound shock with bilateral lung infiltration developed rapidly in 2 days with supportive care and empirical ampicillin/ sulbactam. Bronchoscopy revealed a whitish plaque over bilateral upper bronchi. Biopsy via bronchoscopy revealed moulds with vascular invasion. Culture of bronchial alveolar lavage yielded Aspergillus flavus. The patient died despite amphotericin B treatment, which was started since finding the whitish plaque with bronchoscopy examination. Results and Conclusion Besides to considering capillary leakage syndrome, our case report and literature review alert clinicians that CT and bronchoscopy may help to identify the true pathogen though all cases with concurrent dengue and Aspergillus infections had fatal outcomes.

Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates.

Defining the effects of neuraminidase inhibitors on influenza virus infection may provide important information for the treatment of patients. The effects of neuraminidase inhibitors have been examined using various methods, including viral release from kidney cells. However, the effects of neuraminidase inhibitors on viral release from primary cultures of human tracheal epithelial cells, which retain functions of the original tissues, have not been studied. The effects of neuraminidase inhibitors on the replication of the pandemic influenza virus [A/Sendai-H/N0633/2009 (H1N1) pdm09] and the seasonal influenza virus [A/Sendai-H/216/2009 (H1N1)] that was isolated during the 2008-2009 season were examined. The virus stocks were generated by infecting tracheal cells with the pandemic or seasonal influenza virus. Four types of inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) reduced pandemic viral titers and concentrations of the cytokines interleukin-6 and tumor necrosis factor-α in supernatants and viral RNA in cells. However, oseltamivir did not reduce seasonal viral titers, cytokine concentrations and viral RNA, and the 50% inhibitory concentration (IC50 ) of oseltamivir for neuraminidase activity in the seasonal virus was 300-fold higher than that observed for the pandemic influenza virus. The seasonal influenza virus had an oseltamivir-resistant genotype. The magnitude of the IC50 values of the neuraminidase inhibitors for the seasonal influenza virus was inversely related to the magnitude of the inhibitory effects on viral release. These methods for measuring the release of virus and inflammatory cytokines from primary cultures of human tracheal epithelium may provide useful information regarding the effects of neuraminidase inhibitors on influenza viruses.

With the emerging risks of drug-resistant viruses and pandemic influenza, rapid and accurate detection of influenza viruses and determination of their subtypes is a crucial component of patient management. This study evaluated the performance of the Verigene respiratory virus plus nucleic acid (Verigene RV+) test for the detection of influenza A/B and subtype determination compared it with conventional molecular methods. Nasopharyngeal swabs were collected from 228 patients with influenza-like illness (influenza A (n = 67), 2009-H1N1 (n = 21), influenza B (n = 80), mixed A & B (n = 3), mixed RSV A and influenza (n = 3), and influenza-negative (n = 54)). Patient samples were analyzed by Influenza A/B one-step typing (Seegene, Seoul, Korea), Seeplex RV15 ACE Detection (Seegene), Nanosphere Verigene RV+ assay (Nanosphere, Northbrrook, IL) and virus culture. Out of 228 samples, 109 (47.8%) were positive by culture, and an additional 65 (28.5%) were positive by Seeplex RV15 ACE Detection, Influenza A/B one-step typing or Nanosphere Verigene RV+ assay. In comparison tests with Seeplex RV15 ACE Detection RT-PCR, the sensitivity of the Verigene RV+ kit for detection of the influenza A, 2009-H1N1, influenza B, and mixed A & B was 97.1%, 100%, 100%, and 100%, respectively. The specificity of the Verigene RV+ was 100% for all types. The concordance between Verigene RV+ and Influenza A/B one-step typing for H1, H3, H1/H3 mixed, and 2009-H1N1 was 100% (26/26), 100% (35/35), 100% (4/4), and 100% (21/21), respectively. The Verigene RV+ assay showed acceptable sensitivity and specificity for detection and subtyping of influenza viruses compared with the conventional RT-PCR method. J. Med. Virol. 87: 18–24, 2015. © 2014 Wiley Periodicals, Inc.

Aim Community-acquired pneumonia (CAP) is presumed to be bacterial in origin and empirical antibiotics are almost always given on admission. However, early detection of viral infection is also very important for hospital infection control and timely use of antiviral agents. The present study aimed to compare patients with viral and bacterial pneumonia, and identify independent predictors of viral pneumonia. Methods A prospective cohort study was carried out in a tertiary teaching hospital in a 1-year period. Older patients (aged ≥65 years) were recruited if they were admitted for CAP confirmed by chest radiographs. Results A cohort of 488 patients was analyzed. Infective causes were found in 137 (28.1%) patients. Bacterial, viral and mixed infections were detected in 86 (17.6%), 41 (8.4%) and 10 (2.0%) patients, respectively. Bacteriology was established mostly by sputum culture and virology by nasopharyngeal aspirate (NPA) viral culture. The commonest bacterial isolates were Haemophilus influenzae (31), Pseudomonas aeruginosa (15), Mycobacterium tuberculosis (14), Klebsiella spp. (9) and Streptococcus pneumoniae (6). Influenza A virus (28, 8 were pandemic 2009 A/H1N1 subtype) and respiratory syncytial virus (16) were the most frequent viral causes. Independent predictors of viral pneumonia included nursing home residence (RR 3.056, P = 0.009) and absence of leukocytosis (RR 0.425, P = 0.026). Conclusions All nursing home residents hospitalized for CAP should undergo NPA viral testing because of infection control, early antiviral treatment and discharge planning. We suggest that empirical antiviral agents might be considered for nursing home residents hospitalized for CAP if outbreaks of influenza-like illness are reported in nursing homes. Geriatr Gerontol Int 2013; 13: 949–957.

Adenovirus infection is a significant complication in paediatric AlloSCT recipients with a mortality rate for disseminated adenovirus that may exceed 80%. We sought to determine the incidence, risk factors, and associated outcomes of adenovirus infection in 123 consecutive paediatric AlloSCT recipients. Adenovirus was diagnosed by antigen detection or viral culture, and was defined by isolation of virus with presence of correlating clinical symptoms. The probability of developing adenovirus infection was 12.3% (CI(95) 6.0-18.6). There were no statistically significant differences in probability of adenovirus infection in univariate analysis of risk factors including donor source, use of ATG/Alemtuzumab, ≥ Grade II GVHD, among others (age, diagnosis, conditioning regimen). Probability of overall survival for patients that experienced adenovirus infection was 15.4% vs. 50% for those without adenovirus (P = 0.0286). In multivariate analysis, the most important risk factor for an increased risk of death was adenovirus infection [HR 3.15 (CI(95) 1.6-6.18) P = 0.0009]. In this series of paediatric AlloSCT recipients, the development of adenovirus infection was the leading multivariate predictor of treatment-related mortality. Enhanced surveillance with adenovirus PCR and identification of the risk factors associated with poor outcomes from adenovirus infection may identify patients that may benefit from pre-emptive therapeutic interventions including adenovirus-specific cellular immunotherapies.

P455. 30 Balfour HH Jr, Bean B, Laskin OL et al. Acyclovir halts progression of herpes zoster in immunocompromised patients. N Engl J Med 1983; 308: 1448–1453. 31 Shepp DH, Dandliker PS, Meyers JD. Treatment of varicella-zoster virus infection in severely immunocompromised patients: a randomized comparison r 2011 The Authors r 2011 British HIV Association HIV Medicine (2011), 12 (Suppl. 2), 61–69 6. Herpes viruses 67

The highly pathogenic avian influenza A (H5N1) virus is a virulent virus that causes an acute febrile respiratory disease with high mortality in humans. To gain a better insight of H5N1 viral distributions in infected human tissues, the levels of viral RNA were determined in the autopsy tissues from two patients who were infected with H5N1 virus by using real-time reverse transcription-polymerase chain reaction. In one patient who died on day 6 of the illness, the viral load in the lung was extremely high, whereas the levels of viral RNA in the other organs were more than 6 log lower. In the other patient who died on day 17 of the illness, the viral load was similar in the lung and other organs, and was comparable to the viral load in the extra-pulmonary tissues of the first patient. These results suggested that while the H5N1 virus can cause disseminated infection in humans, the lung is still the major site of viral replication, and viral replication in the lung in the later stages may decrease as a result of the depletion of the available target cells. In addition, the mRNA levels of the tumor necrosis factor-α (TNF-α) were found to be associated with the viral titers.

The development of new technologies has propelled virus discovery efforts forward at an unprecedented pace. As this area of research has become increasingly active, a review of virus discovery efforts over the last ~10 years is warranted. This chapter describes the evolution of sequence-independent PCR methods, panviral microarrays, and mass sequencing in the discovery of novel viruses. It also highlights the viruses discovered by these methods, and the importance and novelties of some of these viruses. Sequence-independent PCR strategies and new technologies such as microarrays and mass sequencing, while not without their own limitations, circumvent many of the limitations associated with traditional virus discovery methods and consequently have revolutionized the process of virus discovery. The discovery in 2001 of a novel human pneumovirus, called human metapneumovirus (HMPV), relied upon a sequence-independent PCR approach in conjunction with viral culture to identify a previously unknown virus. The first application of virus discovery based on cDNA-amplified fragment length polymorphism (VIDISCA) to virus discovery resulted in the identification of HCoV-NL63. DNA microarrays first emerged in the mid-1990s as powerful tools to measure gene expression or genomic content changes in various organisms. Dideoxy sequencing or Sanger sequencing, first described in 1977, has been the dominant DNA sequencing technology used during the last ~30 years. With the advent of new technologies, a shift has occurred such that in many instances, the rate-limiting step is no longer discovery but understanding the biological relevance and impact of newly discovered viruses.

Herpesvirus infections cause morbidity in lung transplant recipients. The study was conducted to investigate the incidence and impact of herpes simplex virus (HSV) and cytomegalovirus (CMV) detection in the respiratory tract (RT) of lung and heart-lung transplant recipients (LTR) during the postoperative phase. In a prospective cohort study, 91 LTR having at least 1 nasopharyngeal swab (NPS) sent for virus diagnostics were monitored for CMV and HSV detection in NPS during their post-transplant hospital stay on cardiothoracic surgery wards (median 4 weeks) by direct immunofluorescence testing for HSV, virus culture, and CMV and HSV polymerase chain reaction (PCR). Bronchoalveolar lavages (BALs) were analyzed with the same protocol except that HSV PCR was only performed on request. Risk factor analysis for the outcome '90-day mortality' was performed. Fifteen LTR had virus detection in NPS (16.5%): 9 had CMV, 5 had HSV, and 1 had both CMV and HSV. Four of 84 LTR had CMV detection in BAL (4.8%). Absence of CMV detection in NPS had a negative predictive value of 98.8% for absence of CMV detection in BAL. HSV DNA detection in NPS, especially if detected within 8 days after transplantation, was associated with 90-day mortality. In conclusion, detection of herpesviruses in the RT was clinically relevant and frequent, despite antiviral prophylaxis.

Summary Background: The diagnosis of parapox virus infections relies primarily on a history of contact with infected animals. The clinical presentation is usually a non-specific necrotic ulcer. The histology may also be non-specific, especially with older lesions. Negative-staining electron microscopy (EM) is a fast and reliable diagnostic tool, but is not widely available. Serological tests and the time-consuming viral culture are also rarely used in Europe. Patients and methods: The diagnostic procedure in two patients with ecthyma contagiosum and milker's nodule using polymerase chain reaction specific for orthopox, parapox and Orf virus is explained. Diagnostics included bacterial culture, viral culture, histology and EM. In addition to these, a polymerase chain reaction (PCR) was performed in both cases. Results: The patient with ecthyma contagiosum was negative for ortho-, parapox-, and orf-virus on PCR, whereas the patient with milker's nodule had a PCR positive for parapoxvirus. Conclusions: PCR is a simple, fast, and standardized method of diagnosis that can distinguish between the subgroups of parapoxviruses. A diagnosis can be made even in cases of ambiguous history or unspecific clinical presentation. The method is limited by the necessity to sample native material or to use neutrally buffered formalin in case of PCR from paraffin material.

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza-specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co-circulating seasonal influenza strains.

Following the clinical diagnosis of the first case of mumps on September 22, 2006 at the University of Virginia (UVA), 52 suspected cases were identified through active surveillance for mumps by the end of December 2006. Samples were collected from 47 students who presented with parotitis despite a documented history of two doses of measles, mumps, and rubella (MMR) vaccine. Six of 47 serum samples (13%) were positive for mumps IgM, and 46/47 specimens were positive for mumps IgG. Endpoint titration of acute phase serum samples from laboratory-confirmed cases did not provide evidence that elevated serum IgG is a consistent marker for infection among cases due to secondary vaccine failure. Buccal swab samples from 39 of the 47 students were tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) and/or viral culture. Mumps virus or mumps RNA was detected in 12 of 39 buccal samples (31%). Genetic analysis of the virus from the outbreak at UVA indicated that the outbreak was not linked to the large mumps outbreak in the Midwestern US that occurred earlier in 2006. Our findings support the use of viral detection to improve laboratory diagnosis of mumps among persons who have received two doses of MMR. J. Med. Virol. 81:1819–1825, 2009. © 2009 Wiley-Liss, Inc.

Polyomavirus BK (BKV) has ebeen identified as the main cause of polyomavirus-associated nephropathy, a major cause of renal allograft failure. Although BKV-associated nephropathy develops in only 2% to 5% of renal transplant recipients, its prognosis when present is very poor, with irreversible graft failure developing in 45% of affected patients. While the use of urine cytology for the detection of decoy cells has been in use for decades, other diagnostic modalities to detect BKV have emerged, including tissue biopsy, polymerase chain reaction, viral culture, and serology. Currently, there is no consensus regarding the laboratory technique best suited for clinical monitoring. This review article will discuss essential and clinical features of polyomavirus, followed by a discussion pertaining to the various diagnostic modalities that contribute to detecting polyomavirus-associated nephropathy. Diagn. Cytopathol. 2009. © 2009 Wiley-Liss, Inc.

Background Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5′- and 3′-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression. Findings Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses. Conclusions These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers.

Sensitive detection of respiratory viruses is important for early diagnosis of infection in patients following hematopoietic cell transplantation (HCT). To evaluate the relative sensitivity of respiratory virus detection in specimens from HCT recipients, we compared the results of conventional and quantitative molecular methods.We tested 688 nasal wash samples collected prospectively from 131 patients during the first 100 days after HCT by viral culture, fluorescent antibody staining (FA), and real-time quantitative reverse transcription-polymerase chain reaction (PCR) assay for detection of respiratory syncytial virus (RSV), influenza virus types A (FluA) and B (FluB), and parainfluenza virus types 1 (PIV1) and 3 (PIV3). Testing for human metapneumovirus (MPV) was performed only by PCR. Data regarding 10 respiratory symptoms were collected with each sample.By any method 37 specimens were positive for a respiratory virus; 34 were positive by PCR, 15 by culture, and 6 by FA. Four specimens were positive by all 3 methods (3 RSV, 1 FluA). One specimen was positive for PIV1, and 2 were positive for rhinovirus by culture alone. Specimens positive by PCR alone included 2 RSV, 2 PIV1, 8 PIV3, and 8 MPV. In 10 specimens positive for RSV, PIV, or influenza virus collected from patients reporting no respiratory symptoms, 9, 4, and 1 specimen were positive by PCR, culture, and FA, respectively. Overall, specimens positive only by PCR had significantly fewer viral copies/mL (mean log(10)=4.32) than specimens positive by both PCR and culture (mean log(10)=5.75; P=0.002) or PCR and FA (mean log(10)=6.83; P0.001).FA testing alone did not detect a significant proportion of respiratory virus-positive samples in HCT recipients, especially in patients with no respiratory symptoms and patients with PIV detection. PCR increased the yield of positive specimens 2 times relative to culture and more than 4 times relative to FA. Detection of respiratory viruses by PCR alone was associated with lower virus quantities and with fewer reported respiratory symptoms compared with concomitant detection by both PCR and conventional methods, indicating that PCR may be important to detect asymptomatic or mildly symptomatic stages of respiratory viral infections.

Objective: To identify and gain an understanding of the influenza viruses circulating in wild birds in Australia. Design: A total of 16,303 swabs and 3782 blood samples were collected and analysed for avian influenza (AI) viruses from 16,420 wild birds in Australia between July 2005 and June 2007. Anseriformes and Charadriiformes were primarily targeted. Procedures: Cloacal, oropharyngeal and faecal (environmental) swabs were tested using polymerase chain reaction (PCR) for the AI type A matrix gene. Positive samples underwent virus culture and subtyping. Serum samples were analysed using a blocking enzyme-linked immunosorbent assay for influenza A virus nucleoprotein. Results: No highly pathogenic AI viruses were identified. However, 164 PCR tests were positive for the AI type A matrix gene, 46 of which were identified to subtype. A total of five viruses were isolated, three of which had a corresponding positive PCR and subtype identification (H3N8, H4N6, H7N6). Low pathogenic AI H5 and/or H7 was present in wild birds in New South Wales, Tasmania, Victoria and Western Australia. Antibodies to influenza A were also detected in 15.0% of the birds sampled. Conclusions: Although low pathogenic AI virus subtypes are currently circulating in Australia, their prevalence is low (1.0% positive PCR). Surveillance activities for AI in wild birds should be continued to provide further epidemiological information about circulating viruses and to identify any changes in subtype prevalence.

The major immediate-early (IE) promoter (MIEP) of cytomegalovirus (CMV) controls the transcription of IE genes, IE1 and IE2, to encode proteins p72 and p86, respectively. Laboratory-based diagnosis is usually required to identify congenital and perinatal CMV disease and to diagnose and monitor viral levels in immunosuppressed hosts. The laboratory techniques commonly employed are the conventional tube and shell vial viral culture techniques, immunological techniques for histopathology, immunohistochemical techniques, antigen and antibody detection, and nucleic acid detection. Acyclovir, which is successfully used against other herpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), is less potent against CMV. As one's understanding of the CMV genome and protein functions further advances, novel vaccine candidates and strategies will evolve that give equivalent or better humoral and cellular immune responses than natural immunity. The availability and versatility of VZV cosmids, which are large DNA fragments of the viral genome that can be recombined to form replication-competent VZV, have allowed targeting of different ORFs with point deletion mutants. A live attenuated vaccine was most effective in persons 60 to 69 years of age, but it also decreased the severity of incident zoster in people 70 years or older. Epstein-Barr virus (EBV) infection in the immunocompromised host is accompanied by the risk of developing lymphoproliferative disease. Using cytotoxic T lymphocyte (CTL) therapy in combination with other treatment modalities for aggressive PT-LPDs that are unresponsive to reduced immunosuppression has successfully induced remission without significant side effects.

This chapter talks about cytopathology of viral infections, and describes the methods used to obtain and prepare cells for cytologic examination. It illustrates characteristic changes encountered in common virus infections. The preparatory methods utilized for the microscopic examination of cytologic specimens can be divided into five categories: direct smears, preparation by cytocentrifugation, membrane filter preparation, monolayer preparation, and cell block preparation. The eye and respiratory, genital, and urinary tracts are locations that readily yield cytologic material for rapid viral diagnosis. Characteristic cytologic changes depend on the cytopathic effect of a virus in infected cells, which need not include all cells of the involved organ. A nonspecific change referred to as ciliocytophthoria is found in various inflammatory diseases of the respiratory tract and, in particular, virus infections. Ciliocytophthoria occurs most frequently with influenza virus, parainfluenza virus, and adenovirus infection but may also occur in bronchiectasis and other nonviral inflammatory conditions. Although many viruses that cause systemic infections have been isolated from urine, those most readily diagnosed by urine cytology are cytomegalovirus (CMV), herpes simplex virus (HSV), and a member of the papovavirus group, designated the BK virus (BKV). Detection of HSV genital tract infection is important in abating the spread of sexually transmitted HSV as well as protecting the neonate from life-threatening infection transmitted to the infant during a vaginal birth. The morphologic changes in conjunctival and corneal cells due to virus infection must be differentiated from the cytologic changes due to chlamydial infections causing trachoma and inclusion conjunctivitis.

The development of methods to amplify nucleic acids has provided a way of identifying and quantifying infectious pathogens in infected pulp and periapical region. Recent studies have detected human herpes virus in periapical pathosis and periodontitis. The aim of this study is to detect the presence or absence of herpes simplex virus, human cytomegalovirus and Epstein Barr virus in an infected pulp. Ten pulp tissue samples from teeth with irreversible pulpitis and eight control samples were subjected to polymerase chain reaction (Perkin – Elmer Gene Amplification System) for detection of human herpes virus. The results of this study did not reveal any human herpes virus in both the control and infected pulp tissue samples. According to this study, human herpes virus may not have an entry through the infected pulp to reach the periapical region and may not be a causative organism in the pulp.

Author(s): Caram, L Brett; Chen, Jodi; Taggart, E William; Hillyard, David R; She, Rosemary; Polage, Christopher R; Twersky, Jack; Schmader, Kenneth; Petti, Cathy A; Woods, Christopher W | Abstract: ObjectivesTo report an outbreak of respiratory synctyial virus (RSV) in a long-term care facility (LTCF) during ongoing routine respiratory illness surveillance.DesignRapid antigen testing, viral culture, direct fluorescent antibody (DFA) testing, and reverse transcriptase polymerase chain reaction (RT-PCR) testing for up to 15 viruses in symptomatic residents and chart review.SettingA 120-bed LTCF.MeasurementsComparison of rapid antigen testing, respiratory viral cultures, and DFA testing and RT-PCR in residents with symptoms of a respiratory tract infection.ResultsTwenty-two of 52 residents developed symptoms of a respiratory tract infection between January 29, 2008, and February 26, 2008. RSV was detected using RT-PCR in seven (32%) of the 22 cases. None of the seven cases had positive RSV rapid antigen testing, and only two had positive culture or DFA results. This outbreak occurred during a time when state wide RSV rates were rapidly declining. One patient was admitted to the hospital during the infection and subsequently died.ConclusionRSV may cause outbreaks in LTCFs that traditional diagnostic methods do not detect. RT-PCR can provide a more timely and accurate diagnosis of outbreaks, which allows for early symptomatic treatment, rational use of antibiotics, and improved infection control.

Viral disease in the central nervous system (CNS) can be classified by pathogenesis. Neurologic disease is frequently categorized as either primary or postinfectious. Primary encephalitis results from direct viral entry into the CNS that produces clinically evident cortical or brain stem dysfunction. Meningitis and encephalitis represent separate clinical entities; however, a continuum exists between these distinct forms of disease. A change in a patient’s clinical condition can reflect disease progression with involvement of different regions of the brain. Epidemiological data in many cases provide clues to the etiology of the illness. Viral meningitis is a relatively benign self-limited illness, and pathological specimens are rarely available for study. The diagnosis of viral meningitis relied on viral culture, and CSF viral culture rates differ based on etiology. The fundamental principle of therapy for viral meningitis lies in the identification of potentially treatable diseases. Similar to the case with viral meningitis, passive reporting systems underestimate the incidence of viral encephalitis. The pathogenesis of encephalitis requires that viruses reach the CNS by hematogenous or neuronal spread. Patients with encephalitis, depending on the etiology and extent of CNS involvement, require treatment tailored to their clinical situation. The approach to a patient with a presumed CNS viral infection must be tailored to the severity and distribution of neurologic involvement. Improvements in ability to diagnose CNS infections will produce a better understanding of the pathogenesis and true extent of CNS viral disease.

The occurrence of viral coinfections in childhood pneumonia has received little attention, probably because suitable detection methods have been lacking. Between November 2004 and October 2006, the presence of 14 respiratory viruses in children aged less than 3 years old with community‐acquired pneumonia were investigated using molecular or immunochromatographic techniques and/or viral culture. A total of 315 children (338 episodes) were included, and hospitalization was required in 178 episodes. At least one virus was detected in 66.9% of the episodes and simultaneous detection of two or more viruses was frequent (27% of the episodes with viral detection). The most frequently detected virus was respiratory syncytial virus (n = 67: 33 subgroup A, 33 subgroup B, 1 not typed), followed by human bocavirus (n = 48), rhinovirus (n = 46), human metapneumovirus (n = 39: 13 genotype A2, 8 B1, 5 B2, 1 A1, 12 not genotyped) and parainfluenza viruses (n = 38: 1 type 1, 3 type 2, 22 type 3, 11 type 4 and 1 not typed). The 14 viruses investigated were found in viral coinfections, which were more frequent in children aged less than 12 months. Except for adenovirus, the incidence of which was low, the percentage of viral coinfection ranged between 28.2% and 68.8%. Children with viral coinfection more frequently required hospital admission than those with single viral infection. It is concluded that viral coinfections are frequent in children aged less than 3 years old with community‐acquired pneumonia and can be a poor prognostic factor. J. Med. Virol. 80:1843–1849, 2008. © 2008 Wiley‐Liss, Inc.

The four viruses herpes simplex virus (HSV), varicella-zoster virus (VZV), human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), discussed in this chapter, are all members of the family Herpesviridae. Each organism is discussed separately in order to better describe the specific details of the pathogenesis, epidemiology, and diagnostic testing. The majority of cases of HSV reactivation occur in the initial few weeks after transplantation, at the time of maximum pharmacologic immunosuppression. As the majority of immunocompromised patients have recurrent infections with HSV, the role of serology in these patients is of limited diagnostic value. Detection of HSV DNA does not require live virus; therefore, specimen quality is not jeopardized by delays in transport time or fluctuations in transport temperature like it would be for viral culture. Tyrosine kinase-negative VZV strains have been identified, and infections caused by the strains should be treated with foscarnet. The immunocompromised states covered in the chapter include patients who have undergone bone marrow and solid organ transplantation. The majority of the literature surrounding the role of HHV-6 in the transplant population regards patients with bone marrow transplantation. A study compared staining for HHV-6 by using immunohistochemistry and in situ hybridization to polymerase chain reaction (PCR) analysis of tonsillar epithelia of nonimmunosuppressed individuals undergoing tonsillectomy in an attempt to determine if lymphoid tissue was a site of latency in the immunocompetent population. Molecular methods of detection for each of these viruses are sensitive, specific, and available with a relatively short turnaround time.

BACKGROUND: A single instance of transfusion-transmitted dengue infection has been reported. The high incidence of dengue in endemic countries, the high proportion of asymptomatic infection, and the median 5-day viremia, however, suggest that transfusion-associated dengue transmission may be more widespread than documented. STUDY DESIGN AND METHODS: The prevalence of dengue virus (DENV) RNA was determined in all blood donations to the American Red Cross in Puerto Rico from September 20 to December 4, 2005, using a specific type of nucleic acid amplification test called transcription-mediated amplification (TMA). TMA-positive donations were defined as those having two repeatedly reactive TMA results. TMA-positive donations were tested by enzyme-linked immunosorbent assay for immunoglobulin M (IgM) antibodies, by reverse transcription–polymerase chain reaction (RT-PCR), and by viral culture. RESULTS: Twelve (0.07%) of 16,521 blood donations tested were TMA-positive. Four were positive by RT-PCR (DENV serotypes 2 and 3). Virus was cultured from 3 of 4 RT-PCR–positive donations. One of the 12 TMA-positive donations was IgM-positive. Only 5 donations remained TMA-positive when diluted 1:16, as is done for routine minipool screening for other transfusion-transmissible viral infections (hepatitis C, human immunodeficiency, West Nile viruses [WNVs]). CONCLUSION: Nearly 1 in 1000 blood donations contained DENV RNA, and virus could be cultured from TMA-positive donations, suggesting a transfusion transmission risk similar to that which existed in the United States for WNV before universal donation screening. Similar to WNV, IgM antibody screening is likely to be ineffective, and some potentially infectious donations will be missed by minipool screening. Transfusion transmission should be considered in patients with dengue after blood transfusion.

IgA-deficient blood donors and their age- and sex-matched controls were compared for the occurrence of complement-fixing antibodies in serum against several viruses. The level in the IgA-deficient persons was slightly higher against several respiratory pathogens (adenoviruses, type B influenza virus, parainfluenza virus, and respiratory syncytial virus) that give rise to localized infections, and against coxsackie B group of viruses. No corresponding difference was observed in mumps, varicella, and cytomegalovirus infections, where viraemia is a characteristic feature, or in Mycoplasma pneumoniae infection.

OBJECTIVE: To determine whether a significant association occurs between the presence of various periodontal diseases and recoverable infectious HIV-1 in the saliva of injecting drug users. DESIGN: Five hundred and fifty-one injecting drug users were recruited from various programs associated with the Beth Israel Medical Center. Examiners were ‘blinded’ to the subject's HIV-1 serostatus. A socio-economic and risk factors’ survey was conducted and a complete oral examination, including periodontal disease indices was performed. Whole saliva and blood were collected for virus culture. MAIN OUTCOME MEASURES: Recovery of infectious HIV-1 in saliva related to presence of periodontal diseases. RESULTS: Those HIV-1 seropositive subjects with periodontal diseases did not differ from those HIV-1 seropositive subjects without periodontal disease in mean age and immune status. Less than 1% of the HIV-1 seropositive subjects had cultivable HIV-1 in their saliva while it was present in 78% of PBMCs and 35% of the sera. There was no significant association between infectious HIV-1 in saliva, serum, or PBMCs and any of the various periodontal diseases. CONCLUSIONS: The presence of periodontal disease in HIV-1 seropositive injecting drug users does not appear to be a potential risk factor for infectious HIV-1 in saliva, probably due to the various anti-viral components of saliva.

Introduction: The purpose of this study was to compare nested polymerase chain reaction (PCR), real-time PCR, and shell vial for the detection of human cytomegalovirus (HCMV) in subgingival samples in periodontitis patients. Methods: A group of 44 patients and 24 individuals without periodontitis were included in the study. A full periodontal examination was conducted in each subject. Gingival crevicular fluid (GCF) was collected by pocket lavage and used for viral culture (shell vial). Additional subgingival samples were obtained with paper points and used for molecular analysis. Nested PCR and real-time PCR were used to detect and quantify HCMV. Student’s t-test and chi-squared test were used to compare groups. The sensitivity and specificity for the tests were calculated on 2 × 2 tables considering the nested PCR as the gold standard. Results: The detection of HCMV was greater using nested PCR than with either real-time PCR or shell vial (P