Virus Isolation In Cell Culture Research Articles

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

Comparative effectiveness of isolation techniques for contemporary Influenza A virus strains circulating in exhibition swine

The current study sought to compare the effectiveness of 2 virus isolation methods for the recovery of contemporary Influenza A virus (FLUAV) strains circulating in swine at agricultural exhibitions. Following the emergence of the influenza A (H1N1)pdm09 virus, increased surveillance of FLUAV strains among swine was recommended for early detection of emerging strains that threaten animal and human health. The increase in genetic drift and genomic reassortment among FLUAV strains infecting swine since 1998 necessitates that detection protocols be periodically validated for contemporary FLUAV strains. During 2009, nasal swabs were collected from 221 clinically healthy pigs at 12 agricultural exhibitions in Ohio. Nasal swabs were tested in parallel for the presence of FLUAV strains using 3 methodologies: 2 passages through Madin-Darby canine kidney (MDCK) cells adapted to serum-free medium (SFM), 2 passages through embryonated chicken eggs (ECEs), and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Of the 221 samples, 40 (18.1%) were positive for FLUAV recovery in MDCK cell culture and 13 (5.9%) were positive in ECEs (P = 0.015). All samples positive in ECEs were also positive in MDCK cell culture. MDCK cell culture virus isolation results were in perfect agreement with results of the real-time RT-PCR. Hemagglutinin and neuraminidase combinations of the recovered isolates were H1N2 and H3N2, which were consistent with FLUAV strains circulating in U.S. pigs. Effectiveness and cost savings justify the use of SFM-adapted MDCK cell culture over ECEs for the recovery of contemporary FLUAV strains from exhibition swine.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

BackgroundFeline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia.FindingsOf the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA.ConclusionsThis study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

DIRECT DETECTION OF ENTEROVIRUS GENOME IN CELL-CULTURE NEGATIVE CEREBROSPINAL FLUID FROM ASEPTIC MENINGITIS CASES IN BRAZIL

Enteroviruses are the main etiologic agents involved in outbreaks and sporadic cases of aseptic meningitis. Viral isolation in permissive cells is a traditional method for diagnosis, but it is time consuming and may be unsuccessful due to low viral titers and because some enteroviruses do not replicate in routine cell cultures. Therefore, molecular techniques are increasingly being used for detection of these agents. In the present work, 267 cerebrospinal fluid (CSF) samples from aseptic meningitis and meningoencephalitis cases, received between 2008 and 2009, were tested by RT-PCR. These clinical samples were previously negative for virus isolation in cell culture. Enterovirus genome was detected in 59 CSF samples (22.1%) and identification was confirmed by partial nucleotide sequencing. Some demographics of the patients included in the study were analyzed. Direct detection of enterovirus genome from CSF is appropriate to increase sensitivity in aseptic meningitis and meningoencephalitis cases. DOI: http://dx.doi.org/10.17525/vrr.v17i1-2.62

Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo (Cacatua moluccensis)

Avian bornaviruses (ABV) have been discovered in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. To date, six ABV genotypes have been described in psittacines. Furthermore, two additional but genetically different ABV genotypes were recognized in non-psittacine birds such as canary birds and wild waterfowl. This remarkable genetic diversity poses a considerable challenge to ABV diagnosis, since polymerase chain reaction (PCR) assays may fail to detect distantly related or as yet unknown genotypes. In this study we investigated the use of virus isolation in cell culture as a strategy for improving ABV diagnosis. We found that the quail fibroblast cell line CEC-32 allows very efficient isolation of ABV from psittacine birds. Isolation of ABV was successful not only from organ samples but also from cloacal and pharyngeal swabs and blood samples collected intra vitam from naturally infected parrots. Importantly, using this experimental approach we managed to isolate a new ABV genotype, termed ABV-7, from a salmon-crested cockatoo (Cacatua moluccensis). Phylogenetic analysis showed that ABV-7 is most closely related to the psittacine genotypes ABV-1, -2, -3, and -4 and clearly distinct from genotypes ABV-5 and -6. Our successful identification of ABV-7 emphasizes the necessity to consider the high genetic diversity when trying to diagnose ABV infections with high reliability and further shows that classical virus isolation may represent a useful diagnostic option, particularly for the detection of new ABV genotypes.

Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia

Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution.

Predictive factors and viral genetic diversity for viral hemorrhagic septicemia virus infection in Lake Ontario and the St. Lawrence River

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of a devastating disease in fish, viral hemorrhagic septicemia (VHS), which has recently spread to the Laurentian Great Lakes. In this paper, we report the results of infection surveillance conducted during 2009 for VHSV with a focus on yellow perch (Perca flavescens) and round goby (Neogobius melanostomus). We collected 1928 fish representing eight species at nine sites in Lake Ontario and the St. Lawrence River and found fish positive for VHSV by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) at every site. Prevalence of virus-positive fish at individual sites ranged from 0.7% to 27.5%. Yellow perch were significantly less likely to test positive than round goby in this study. In logistic regression models, maturity was a significant predictor of testing positive in yellow perch, but no evaluated factor was a significant predictor of testing positive in round goby. Virus isolation in cell culture was successful in seven out of 37 high titer fish samples, and partial glycoprotein gene sequences obtained from these virus isolates showed an increase in genetic diversity relative to the previously known diversity of Laurentian Great Lakes VHSV genotype IVb. These results highlight the importance of continued movement restrictions and surveillance for VHSV in wild fish in the Laurentian Great Lakes as well as suggest future work to further elucidate the infection dynamics of this virus in wild fish populations.

Molecular Characterization and Phylogenetic Study of Coxsackievirus A24v Causing Outbreaks of Acute Hemorrhagic Conjunctivitis (AHC) in Brazil

BackgroundCoxsackievirus A24 variant (CA24v) is the most prevalent viral pathogen associated with acute hemorrhagic conjunctivitis (AHC) outbreaks. Sixteen years after its first outbreak in Brazil, this agent reemerged in 2003 in Brazil, spread to nearly all states and caused outbreaks until 2005. In 2009, a new outbreak occurred in the northeast region of the country. In this study, we performed a viral isolation in cell culture and characterized clinical samples collected from patients presenting symptoms during the outbreak of 2005 in Vitória, Espírito Santo State (ES) and the outbreak of 2009 in Recife, Pernambuco State (PE). We also performed a phylogenetic analysis of worldwide strains and all meaningful Brazilian isolates since 2003.Methods and FindingsSterile cotton swabs were used to collect eye discharges, and all 210 clinical samples were used to inoculate cell cultures. Cytopathic effects in HEp-2 cells were seen in 58 of 180 (32%) samples from Vitória and 3 of 30 (10%) samples from Recife. Phylogenetic analysis based on a fragment of the VP1 and 3C gene revealed that the CA24v causing outbreaks in Brazil during the years 2003, 2004 and 2005 evolved from Asian isolates that had caused the South Korean outbreak of AHC during the summer of 2002. However, the 2009 outbreak of AHC in Pernambuco was originated from the reintroduction of a new CA24v strain that was circulating during 2007 in Asia, where CA24v outbreaks has been continuously reported since 1970.ConclusionsThis study is the first phylogenetic analysis of AHC outbreaks caused by CA24v in Brazil. The results showed that Asian strains of CA24v were responsible for the outbreaks since 1987 and were independently introduced to Brazil in 2003 and 2009. Phylogenetic analysis of complete VP1 gene is a useful tool for studying the epidemiology of enteroviruses associated with outbreaks.

DIAGNOSIS AND CONTROL OF AUJESZKY’S DISEASE IN PIGS

Aujeszky’s disease (Morbus Aujeszky, MA) is an infectious viral disease caused by swine alpha-herpes virus 1. The disease may be found in diff erent animal species, both domestic and wild, except in primates and humans. Pigs are a natural reservoir of the virus. Th ey are the only species that can survive the infection, but with a possibility of subclinical and latent infection. The main way of spreading the infection is through introduction of subclinically infected pigs into a herd. On the other hand, if the case of stress (transport, capture, dust) reactivation of latent infection and excretion of the virulent virus occurs. Th e aim of this study was to present the methods used in diagnosis and diff erential diagnosis. Th e material for examination originated from two pig farms with increased mortality of suckling piglets. The epizootic and clinical examination were used as well as pathomorpohological examination, standard laboratory methods for determining the presence of aerobic and anaerobic bacteria, virus isolation in cell culture, serum neutralization test for determining the titer of specifi c antibodies against MA and microbiological testing of feed samples. Th e cases of MA on the pig farm where immunoprophylaxis against MA was not carried out, and the breeding animals were purchased from pig farms with unknown health status, causes great direct losses, like for example mortality of piglets, abortion, as well as indirect losses, like growth retardation and high cost of disease control.

Genetic differentiation of infected from vaccinated animals after implementation of an emergency vaccination strategy against classical swine fever in wild boar

Oral emergency vaccination against classical swine fever is a powerful tool to control disease outbreaks among European wild boar and thus to safeguard domestic pigs in affected regions. In the past, when virus detection was mainly done using virus isolation in cell culture or antigen enzyme-linked immunosorbent assays, modified live vaccine strains like C-strain “Riems”, were barely detectable after oral vaccination campaigns. Nowadays, the use of highly sensitive molecular techniques has given rise to an increase in vaccine virus detections. This was also the case during the 2009 outbreak among German wild boar and the subsequent vaccination campaigns. To guarantee a rapid differentiation of truly infected from C-strain vaccinated animals, a combination of differentiating multiplex rRT-PCR assays with partial sequencing was implemented. Here, we report on the rational and use of this approach and the lessons learned during execution. It was shown that positive results in the recently developed vaccine strain (genotype) specific rRT-PCR assay can be taken as almost evidentiary whereas negative results should be confirmed by partial sequencing. Thus, combination of multiplex rRT-PCR assays as a first line differentiation with partial sequencing can be recommended for a genetic DIVA strategy in areas with oral vaccination against classical swine fever in wild boars.

Enterovirusdiagnostik bei aseptischer Meningitis im Kindesalter mit Liquor und/oder Stuhl: Zellkulturgestützte Virusisolierung oder PCR?

Aseptic meningitis in children is most frequently caused by enteroviruses. In clinical setting there arises the question, whether stool samples or cerebrospinal fluid (CSF) are better suited for laboratory diagnosis. Between 06/2006 and 03/2010 a total number of 613 specimens (376 CSF [61.4%] and 237 stool [38.6%]) of 613 children (270 female [44.1%] and 343 [55.9%] male) aged 22 days to 15 years were collected and investigated by enterovirus-PCR and by virus isolation in cell culture, respectively. Enterovirus was detected in 35.4% (217/613) of the samples by cell culture and/or PCR. In total, specimens of CSF were significantly (p<0.001) more often positive (59.4%, n=129) for enterovirus than stool (40.6%; n=88). Concerning stool, PCR gained no significant advantage when compared to cell culture (35.4% vs. 27.8%; p=0.08), However, virus isolation in cell culture using CSF samples succeeded significantly PCR (30.1% vs. 13.8%; p<0.001). Because of its not-invasive sampling, stool is the preferred specimen in suspicion of aseptic meningitis in pediatric clinic. If only stool is available, PCR as well as cell culture are valuable; on contrary, if CSF is available cell culture is significantly superior to PCR in attaining a positive result.

Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1

Two important components of control programs for Bovine herpesvirus type 1 (BoHV-1), a major pathogen of cattle, are the detection of outbreaks and vaccination with glycoprotein E (gE)-deleted marker vaccines. In addition to serology, rapid and accurate investigation of BoHV-1 and genetic differentiation of vaccine and wild-type strains are also important methods. Therefore, a triplex quantitative real-time polymerase chain reaction (qPCR) for testing BoHV-1 was developed. Apart from a BoHV-1 specific glycoprotein D (gD) assay, a gE-specific system for differentiation between wild-type BoHV-1 and gE-deleted vaccine strains was established. Finally, an internal control, based on the beta-actin gene was introduced successfully completing the multiplex system.The triplex BoHV-1-qPCR has an analytical sensitivity of less than 10 genome copies per reaction, and the diagnostic sensitivity was equal to or even greater than that of the ‘gold standard’ method of virus isolation in cell culture. A series of reference strains, including gE-deleted BoHV-1 and field isolates were detected reliably. For validation of the specificity of the test, nasal swabs, semen and different organ material from cattle, negative for BoHV-1, and genetically related herpesvirus strains were examined.The new multiplex BoHV-1-specific qPCR system allows highly sensitive and rapid genetic detection and differentiation of BoHV-1 and will be a valuable method for the control of BoHV-1 infection.

Genital Herpes / Genitalni herpes

Abstract Genital herpes is a chronic, nearly always active herpes simplex virus (HSV) infection of sacral ganglia, that may appear bilaterally and in more ganglia than previously thought. It represents one of the most prevalent sexually transmitted infections, and the most frequent cause of genital ulcer disease in the general populations of developed countries. It is caused by HSV type-2 (HSV-2) in 60-80% of cases, with HSV-1 infections causing the remainder. Genital herpes caused by HSV-1 is on the rise. Since genital HSV-1 infections have higher risk for transmission from mother to infant during delivery than HSV-2, they account for 30% of all cases of neonatal herpes. Serological studies have found prevalence of HSV-2 in the general population of developed contries to be up to 25%. Thirty years ago, herpes was defined as “Today’s Scarlet Letter”in the absence of reliable serological tests and highly effective medications, for diagnosis and treatment of genital herpes. In 2000, apart from virus isolation in cell culture (70% sensitivity), that has long been regarded as the diagnostic gold standard, type specific serological tests and higly effective antiviral agents have evolved. However, the following questions were raised: should serological testing be routinely recommended in asymptomatic patients; can antiviral therapy reduce asymptomatic shedding of the virus; can antiviral therapy reduce sexual transmission of infection; can antiviral therapy reduce acquisitation of viral copathogens, such as human immunodeficiency virus (HIV)? Now, ten years later, we know the answers. Type specific HSV DNA detection by real-time PCR assays (100% sensitivity) are tests of choice for every person with recurrent genital ulcers lasting more than 4 days, and must be available in those laboratories currently performing a significant number of PCR tests for different purposes. Type specific IgG serology assays are indicated in all asymptomatic persons who are at increased risk for HSV infection. In sexually active patients experiencing ≥ 6 recurrences per year, daily supressive dose of acyclovir, valacyclovir or famciclovir should be discontinued after a maximum of a year of continuous antiviral therapy in order to reassess recurrence frequency. If necessary, the therapy should be restarted after at least two recurrences. With such expansive diagnostic possibilities and more aggressive therapeutic approaches, we can define genital herpes not as a “Scarlet Letter”, but as a “widespread untoward consequence of human sexuality”.

Susceptibility of hatchery-reared snubnose pompano Trachinotus blochii to natural betanodavirus infection and their immune responses to the inactivated causative virus

Mass mortality of snubnose pompano Trachinotus blochii fry exhibiting dark coloration, anorexia, and abnormal swimming behavior was recently documented at the hatchery of the Aquaculture Department of the Southeast Asian Fisheries Development Center, Philippines. Samples of brain tissues were collected from affected fish and processed for RT-PCR amplification and virus isolation in cell culture. Infected E-11 cells exhibited cytopathic effect characteristic of betanodavirus. Histopathology of moribund fish showed pronounced vacuolations in the brain, spinal cord, and retina. An RT-PCR product of approximately 430bp was amplified from the culture supernatant of betanodavirus-infected E-11 cells and sequenced. Sequencing of the T4 region of the coat protein gene (RNA 2) revealed clustering of the isolated virus within the red-spotted grouper nervous necrosis virus type. The pathogenicity of the isolated betanodavirus in healthy pompano juveniles and fry was determined via intramuscular injection and immersion challenges, respectively. Higher mortality rates were obtained in challenged fish compared with the controls. An inactivated vaccine was subsequently prepared by treating the clarified betanodavirus with formalin. Pompano juveniles intraperitoneally injected with the inactivated vaccine exhibited neutralizing antibodies from days 15 (mean titer 1:240) to 125 (1:560) with the highest titer noted at day 64 (1:2240) post-vaccination. Additionally, pompano fry bath-vaccinated and consequently bath-challenged with betanodavirus at day 35 post-vaccination showed higher survival rate compared with the control, indicating the potential of the inactivated betanodavirus vaccine against VNN in pompano fry and juveniles.

Zoonotic Infections of Buffalopox in India

Four outbreaks of buffalopox in domestic buffaloes, with considerable mortality with high case fatality rates in young buffalo calves and high morbidity with significant productivity loss in terms of reduction in milk yield in adult animals along with severe zoonotic infection in milk attendants were recorded at various places in India, during 2006-2008. In buffaloes, the pox lesions were confined to udder and teats of the majority of the affected animals, and in few animals the lesions were appeared on the hindquarters, indicating generalized infection. The overall disease morbidity, mortality and case fatality rate were 6.8%, 0.7% and 11.4% respectively. Milkers developed pox-like lesions on the hands, forearms and forehead accompanied by fever, axillary lymphadenopathy and general malaise. The causative agent of the outbreaks, buffalopox virus (BPXV), was confirmed upon virus isolation in cell culture, electron microscopy, A-type inclusion (ATI) and ankyrin repeat protein (C18L) gene-specific polymerase chain reactions (PCR). Further, sequence analysis of the BPXV isolates from human and buffalo showed more identity of ATI and C18L genes sequences with that of other orthopoxviruses at nucleotide and amino acid levels and confirmed a close relationship of BPXV with Vaccinia virus (VACV) or VACV-like viruses. Considering the zoonotic impact and productivity losses of buffalopox infection, the control measures are imperative in curtailing economic and public health impact of the disease.

Detection of Infectious Pancreatic Necrosis Virus in Subclinically Infected Atlantic Salmon by Virus Isolation in Cell Culture or Real-Time Reverse Transcription Polymerase Chain Reaction: Influence of Sample Preservation and Storage

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at -80°C for 1month and storage of tissue homogenized in transport medium (TM) at +4°C for 1week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at -80°C after homogenization in TM.

Virological diagnosis of dengue fever in Jeddah, Saudi Arabia: Comparison between RT-PCR and virus isolation in cell culture

A total of 233 serum samples were collected from patients presenting to King Abdulaziz University Hospital with suspected cases of Dengue Fever (DF) from 2006 to 2008. Dengue virus was successfully isolated from 70 samples by culture on C6/36 and LLC-MK2 cells; it was then detected by indirect immunofluorescence assay (IFA). The cytopathic effect (CPE) of dengue virus on C6/36 appeared in most of the samples within 1-4 days post-inoculation comparing to 7-12 days on LLC-MK2 cells, and this was characterized by the ability to induce syncytia and multinucleated giant cells. On the other hand, by using RT-PCR technique, 87 (37.3%) samples were positive. All 70 (30.4%) samples with positive cell culture results were detectable by RT-PCR in addition to 17 culture-negative samples were RT-PCR positive. Dengue virus type 1 (DENV-1) was the dominant serotype followed by DENV- 3 and DENV-2, while DENV-4 was not detected in tested samples. These results indicate that DENV-RNA detection by RT-PCR is more sensitive than virus isolation. We suggest that the high sensitivity coupled with the turnaround time, have made the RT-PCR a better choice as a routine test for DENV diagnosis. Key words: Dengue virus, Dengue fever, RT-PCR, IIF, Saudi Arabia, Jeddah, Dengue diagnosis, virus isolation, virus replication, C6/36, LLCMK-2.

Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines

A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research.

Novel Swine—Origin Influenza A: The 2009 H1N1 Influenza Virus

During April-May 2009, a novel H1N1 influenza virus was determined to be the cause of influenza outbreaks in North America. By June 2009, widespread infections were recorded all over the world and an ongoing pandemic was declared. The clinical manifestations ranged from a self limited illness to fatal disease. Current clinical data suggest that the highest infection rates and complications occur in children and young adults. In contrast to seasonal influenza, the rates of hospitalization and death in adults 65 years or older were low. Risk factors for severe disease are similar to those of seasonal influenza and include diverse medical conditions. However, pregnant women and children with neurodevelopmental disorders or chronic pulmonary conditions are at highest risk of developing severe disease. Rapid antigen detection tests have variable and suboptimal sensitivity for diagnosis of novel H1N1 influenza. Diagnosis is confirmed by real-time reverse transcriptase polymerase chain reaction or by virus isolation in cell culture. Treatment is recommended with oral oseltamivir or inhaled zanamavir for patients who are at risk of complications as well as those who are worsening clinically or have evidence of lower respiratory tract infection. Treatment with intravenous peramivir can be used in special situations when oral or inhaled antiviral therapy is not tolerated or considered inadequate. Inactivated and live attenuated vaccines are available. Current vaccination recommendations and infection control measures are discussed.

Herpesvírus bovinos (BoHV-1.1 e BoHV-1.2b) em forma infecciosa em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul

Verificou-se a incidência de herpesvírus bovinos (BoHVs) em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul. Para tanto, amostras coletadas durante dois anos (n=70) foram submetidas ao isolamento viral em cultivos celulares. Os BoHVs foram isolados em dois (2,9%) encéfalos. Após serem submetidas à caracterização antigênica e molecular, as amostras foram subtipadas como BoHV-1.1 e BoHV-1.2b. A BoHV-1.1 foi isolada de um encéfalo que foi também positivo para raiva. O vírus da raiva foi identificado em 11 amostras (15,7%). Estes achados revelam que a incidência de BoHVs em forma infecciosa em bovinos com encefalite foi baixa, embora represente 16,7% (2/12) dos encéfalos nos quais um agente viral foi identificado. Tal fato confirma a já reportada associação entre BoHV-1 e encefalites. Esse é o primeiro relato da ocorrência de BoHV-1.2b, um subtipo considerado menos patogênico, em um caso de doença neurológica em bovinos.