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Detection of HHV-6 genotypes by in situ hybridization with variant-specific oligonucleotide probes

Arivananthan M, Yadav M, Kumar S

J Virol Methods, 1997 Jun;66(1):5-14.
PMID: 9220385

Human herpesvirus-6 exists in two forms, HHV-6A which has not been clearly associated with any disease, and HHV-6B, the causative agent of exanthem subitum. The two variants have been distinguished by techniques such as dot blotting and restriction fragment length polymorphism of PCR products. This study aims to establish the prevalence of HHV-6A and HHV-6B in carcinoma tissues using variant-specific oligonucleotide probes. A total of 73 archived carcinoma biopsies from the oral, salivary gland, larynx, breast and cervix were obtained with seven histologically normal controls. In situ hybridization was carried out with nonradioactively labelled variant-specific probes. Samples that hybridized with both variant A and B probes were subjected further to nested PCR and digested with HindIII to distinguish the variants. A hybridization signal was observed in 76.2% of oral carcinoma tissue and 75.0% of salivary gland carcinoma tissue. In contrast, only 33.3% of cervical carcinoma tissue were positive for HHV-6 DNA. A hybridization signal was noted in all 4 laryngeal carcinoma tissues studied. However, the 10 breast carcinoma tissues studied were negative, as was the histologically normal tissue. The virus possesses tumourigenic potential and demonstrates virus transactivating properties. The frequency of HHV-6 variants in certain tumours suggest a cofactorial role in multistep carcinogenesis. While PCR amplifies selectively the predominant variant in a sample, this was not seen by in situ hybridization. The in situ hybridization technique allowed the localization of both HHV-6A and HHV-6B in the nuclei of transformed regions.
A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus

Bidawid S, Malik N, Adegbunrin O, Sattar SA, Farber JM

J Virol Methods, 2003 Feb;107(2):163-7.
PMID: 12505630

Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).
Rapid concentration and detection of hepatitis A virus from lettuce and strawberries

Bidawid S, Farber JM, Sattar SA

J Virol Methods, 2000 Aug;88(2):175-85.
PMID: 10960705

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.
Development of a dot enzyme immunoassay for dengue 3: a sensitive method for the detection of antidengue antibodies

Cardosa MJ, Hooi TP, Shaari NS

J Virol Methods, 1988 Oct;22(1):81-8.
PMID: 3058737

Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.
The influence of antibody levels in dengue diagnosis by polymerase chain reaction

Chan SY, Kautner IM, Lam SK

J Virol Methods, 1994 Oct;49(3):315-22.
PMID: 7868649

The potential of RT-PCR to rapidly diagnose dengue infections from both acute and convalescent phase patients' sera was evaluated. The RNA extraction method involved binding of the viral RNA to silica particles in the presence of high concentration of guanidine thiocyanate. The protocol that was established was sensitive enough to detect 40 plaque forming units per 100 microliter of serum and results could be obtained within one day. Results from this study indicate that clinical samples should be collected in the early acute phase of illness when anti-dengue antibodies were undetectable or of low titres to ensure a more reliable diagnosis.
Screening for nasopharyngeal carcinoma with an ELISA using the Epstein-Barr virus nuclear antigen, EBNA 1: a complementary test to the IgA/VCA immunofluorescence assay

Cheng HM, Foong YT, Mathew A, Sam CK, Dillner J, Prasad U

J Virol Methods, 1993 Apr;42(1):45-51.
PMID: 7686558

An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.
Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication

Chin VK, Atika Aziz NA, Hudu SA, Harmal NS, Syahrilnizam A, Jalilian FA, et al.

J Virol Methods, 2016 10;236:117-125.
PMID: 27432115 DOI: 10.1016/j.jviromet.2016.07.012

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection.
Characterisation of mouse monoclonal antibodies targeting linear epitopes on Chikungunya virus E2 glycoprotein

Chua CL, Chan YF, Sam IC

J Virol Methods, 2014 Jan;195:126-33.
PMID: 24134938 DOI: 10.1016/j.jviromet.2013.10.015

Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which has recently re-emerged globally and poses a major threat to public health. Infection leads to severe arthralgia, and disease management remains supportive in the absence of vaccines and anti-viral interventions. The high specificities of monoclonal antibodies (mAbs) have been exploited in immunodiagnostics and immunotherapy in recent decades. In this study, eight different clones of mAbs were generated and characterised. These mAbs targeted the linear epitopes on the CHIKV E2 envelope glycoprotein, which is the major target antigen during infection. All the mAbs showed binding activity against the purified CHIKV virion or recombinant E2 when analysed by immunofluorescence, ELISA and Western blot. The epitopes of each mAb were mapped by overlapping synthetic peptide-based ELISA. The epitopes are distributed at different functional domains of E2 glycoprotein, namely at domain A, junctions of β-ribbons with domains A and B, and domain C. Alignment of mAb epitope sequences revealed that some are well-conserved within different genotypes of CHIKV, while some are identical to and likely to cross-react with the closely-related alphavirus O'nyong-nyong virus. These mAbs with their mapped epitopes are useful for the development of diagnostic or research tools, including immunofluorescence, ELISA and Western blot.
A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies

Crameri G, Wang LF, Morrissy C, White J, Eaton BT

J Virol Methods, 2002 Jan;99(1-2):41-51.
PMID: 11684302

Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.
Fulltext Diversity of respiratory viruses detected among hospitalized children with acute lower respiratory tract infections at Hospital Serdang, Malaysia

Etemadi MR, Jalilian FA, Othman N, Lye MS, Ansari S, Yubbu P, et al.

J Virol Methods, 2019 07;269:1-6.
PMID: 30910688 DOI: 10.1016/j.jviromet.2019.03.013

BACKGROUND: The role of respiratory viruses as the major cause of acute lower respiratory tract infections (ALRTIs) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. The aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with ALRTIs.
METHODS: The cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (RSV), human metapneumovirus (HMPV), influenza virus A and B (IFV-A and B), parainfluenzavirus 1, 2, 3 and 4 (PIV 1, 2, 3 and 4), human rhinoviruses (HRV), human enterovirus (HEV), human coronaviruses (HCoV) 229E and OC43, human bocavirus (HBoV) and human adenovirus (HAdV) in hospitalized children with ALRTIs, at Hospital Serdang, Malaysia, from June 16 to December 21, 2009. The study was also designed in part to assess the performance of the conventional methods against molecular methods.
RESULTS: Viral pathogens were detected in 158 (95.8%) of the patients. Single virus infections were detected in 114 (67.9%) patients; 46 (27.9%) were co-infected with different viruses including double-virus infections in 37 (22.4%) and triple-virus infections in 9 (5.5%) cases. Approximately 70% of samples were found to be positive using conventional methods compared with 96% using molecular methods. A wide range of respiratory viruses were detected in the study. There was a high prevalence of RSV (50.3%) infections, particularly group B viruses. Other etiological agents including HAdV, HMPV, IFV-A, PIV 1-3, HBoV, HCoV-OC43 and HEV were detected in 14.5, 9.6, 9.1, 4.8, 3.6, 2.4 and 1.8 percent of the samples, respectively.
CONCLUSION: Our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of ARTIs in hospitalized children. This is the first report of HMPV infections in Malaysia.
Fulltext SARS-CoV-2 multiplex RT-PCR to detect variants of concern (VOCs) in Malaysia, between January to May 2021

Fu JYL, Chong YM, Sam IC, Chan YF

J Virol Methods, 2022 Mar;301:114462.
PMID: 35026305 DOI: 10.1016/j.jviromet.2022.114462

Emerging SARS-CoV-2 variants of concern (VOC) have been associated with enhanced transmissibility and immune escape. Next-generation sequencing (NGS) of the whole genome is the gold standard for variant identification for surveillance but is time-consuming and costly. Rapid and cost-effective assays that detect SARS-CoV-2 variants are needed. We evaluated Allplex SARS-CoV-2 Master Assay and Variants I Assay to detect HV69/70 deletion, Y144 deletion, E484K, N501Y, and P681H spike mutations in 248 positive samples collected in Kuala Lumpur, Malaysia, between January and May 2021. Spike variants were detected in 78/248 (31.5 %), comprising 60 VOC B.1.351 (beta) and 18 B.1.1.7 (alpha). With NGS as reference for 115 samples, the sensitivity for detecting the spike mutations was 98.7 % with the Master Assay and 100 % with the Variants I Assay. The emergence of beta variants correlated with increasing COVID-19 infections in Malaysia. The prevalence of alpha VOC and lineage B.1.466.2 was low. These assays detect mutations present in alpha, beta and gamma VOCs. Of the VOCs which have subsequently emerged, the assays should detect omicron (B.1.1.529) but not B.1.617.2 (delta). In conclusion, spike variant PCR assays can be used to rapidly monitor selected SARS-CoV-2 VOCs in resource-limited settings, but require updates as new variants emerge.
Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria

Goh ZH, Tan SG, Bhassu S, Tan WS

J Virol Methods, 2011 Jul;175(1):74-9.
PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
Specific detection of Nipah virus using real-time RT-PCR (TaqMan)

Guillaume V, Lefeuvre A, Faure C, Marianneau P, Buckland R, Lam SK, et al.

J Virol Methods, 2004 Sep 15;120(2):229-37.
PMID: 15288966

Nipah and Hendra viruses belong to the novel Henipavirus genus of the Paramyxoviridae family. Its zoonotic circulation in bats and recent emergence in Malaysia with fatal consequences for humans that were in close contact with infected pigs, has made the reinforcement of epidemiological and clinical surveillance systems a priority. In this study, TaqMan RT-PCR of the Nipah nucleoprotein has been developed so that Nipah virus RNA in field specimens or laboratory material can be characterized rapidly and specifically and quantitated. The linearity of the standard curve allowed quantification of 10(3) to 10(9) RNA transcripts. The sensitivity of the test was close to 1 pfu. The kinetics of Nipah virus production in Vero cells was monitored by the determination of infectious virus particles in the supernatant fluid and by quantitation of the viral RNA. Approximately, 1000 RNA molecules were detected per virion, suggesting the presence of many non-infectious particles, similar to other RNA viruses. TaqMan real-time RT-PCR failed to detect Hendra virus DNA. Importantly, the method was able to detect virus despite a similar ratio in viremic sera from hamsters infected with Nipah virus. This standardized technique is sensitive and reliable and allows rapid detection and quantitation of Nipah RNA in both field and experimental materials used for the surveillance and specific diagnosis of Nipah virus.
Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein

Kaku Y, Park ES, Noguchi A, Inoue S, Lunt R, Malbas FF, et al.

J Virol Methods, 2019 07;269:83-87.
PMID: 30954461 DOI: 10.1016/j.jviromet.2019.03.009

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.
A solid-phase blocking ELISA for detection of antibodies to Nipah virus

Kashiwazaki Y, Na YN, Tanimura N, Imada T

J Virol Methods, 2004 Nov;121(2):259-61.
PMID: 15381364

A monoclonal antibody (MAb) based solid-phase blocking ELISA was developed for detection of antibodies to Nipah virus. The ELISA was designed to detect remaining antigens on the plate with anti-Nipah MAb conjugate after the reaction with sample serum, and enabled simple procedure, detection of neutralizing antibody to Nipah virus, and application of samples from different animal species. Forty of 200 swine reference sera examined were positive by the ELISA, of which thirty seven were found positive by serum neutralization test. Sera from a total of 131 fruit bats captured in Malaysia were also tested and all found negative by the both tests. It is considered that the solid-phase blocking ELISA can be used as a screening test for Nipah virus infection followed by the serum neutralization test as confirmatory test.
Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus

Kho CL, Mohd-Azmi ML, Arshad SS, Yusoff K

J Virol Methods, 2000 Apr;86(1):71-83.
PMID: 10713378

A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.
Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus

Kong LL, Omar AR, Hair Bejo M, Ideris A, Tan SW

J Virol Methods, 2009 Nov;161(2):271-9.
PMID: 19591873 DOI: 10.1016/j.jviromet.2009.06.023

A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.
Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

Kong YY, Thay CH, Tin TC, Devi S

J Virol Methods, 2006 Dec;138(1-2):123-30.
PMID: 17000012 DOI: 10.1016/j.jviromet.2006.08.003

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.
Fulltext Evaluation of a commercial dengue NS1 antigen-capture ELISA for laboratory diagnosis of acute dengue virus infection

Kumarasamy V, Wahab AH, Chua SK, Hassan Z, Chem YK, Mohamad M, et al.

J Virol Methods, 2007 Mar;140(1-2):75-9.
PMID: 17140671

A commercial dengue NS1 antigen-capture ELISA was evaluated to demonstrate its potential application for early laboratory diagnosis of acute dengue virus infection. Dengue virus NS1 antigen was detected in 199 of 213 acute serum samples from patients with laboratory confirmation of acute dengue virus infection but none of the 354 healthy blood donors' serum specimens. The dengue NS1 antigen-capture ELISA gave an overall sensitivity of 93.4% (199/213) and a specificity of 100% (354/354). The sensitivity was significantly higher in acute primary dengue (97.3%) than in acute secondary dengue (70.0%). The positive predictive value of the dengue NS1 antigen-capture ELISA was 100% and negative predictive value was 97.3%. Comparatively, virus isolation gave an overall positive isolation rate of 68.1% with a positive isolation rate of 73.9 and 31.0% for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 66.7% with a detection rate of 65.2 and 75.9% for acute primary dengue and acute secondary dengue, respectively. The results indicate that the commercial dengue NS1 antigen-capture ELISA may be superior to virus isolation and RT-PCR for the laboratory diagnosis of acute dengue infection based on a single serum sample.
Identification of the cell binding domain in Nipah virus G glycoprotein using a phage display system

Lam CW, AbuBakar S, Chang LY

J Virol Methods, 2017 05;243:1-9.
PMID: 28082163 DOI: 10.1016/j.jviromet.2017.01.004

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with unusual broad host tropism and is designated as a Category C pathogen by the U.S. National Institute of Allergy and Infectious Diseases. NiV infection is initiated after binding of the viral G glycoprotein to the host cell receptor. The aim of this study was to map the NiV G glycoprotein cell binding domain using a phage display system. The NiV G extracellular domain was truncated and displayed as attachment proteins on M13 phage g3p minor coat protein. The binding efficiency of recombinant phages displaying different regions of NiV G to mammalian cells was evaluated. Results showed that regions of NiV G consisting of amino acids 396-602 (recombinant phage G4) and 498-602 (recombinant phage G5) demonstrated the highest binding to both Vero (5.5×103 cfu/ml and 5.6×103 cfu/ml) and THP-1 cells (3.5×103 cfu/ml and 2.9×103 cfu/ml). However, the binding of both of these recombinant phages to THP-1 cells was significantly lower than to Vero cells, and this could be due to the lack of primary host cell receptor expression on THP-1 cells. Furthermore, the binding between these two recombinant phages was competitive suggesting that there was a common host cell attachment site. This study employed an approach that is suitable for use in a biosafety level 2 containment laboratory without the need to use live virus to show that NiV G amino acids 498-602 play an important role for attachment to host cells.

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