Determining effectiveness of rotavirus vaccine by immunochromatography and reverse transcriptase polymerase chain reaction: A comparison.

INTRODUCTION: Because of the large animal reservoirs and reassortment capacity of rotaviruses (RVs) that pose the possibilities of waning the effectiveness of RV-vaccines, it remains essential to monitor vaccine effectiveness (VE) regularly. Although reverse transcription polymerase chain reaction (RT-PCR) remains sensitive for RV detection, physicians, especially in Japan, frequently use immunochromatography (IC)-based kits for RV diagnosis. Recently, IC is being used to calculate VE also. Herein, we investigated the validity of VEs determined by IC compared to that by RT-PCR during an outbreak in Shizuoka Prefecture, Japan. METHODS: RVs in the stool or rectal swabs from children with acute gastroenteritis (AGE) were tested first by IC in the clinic and then by RT-PCR in the laboratory. A test-negative study design was used to examine VE. RESULTS: Although the specificity of IC assay revealed 100%, its sensitivity remained weaker (67%) than that of RT-PCR that increased up to 88% depending on disease severity. VE assessed by IC remained stronger than that by RT-PCR: 79% (95% CI: 39-93%) by IC, and 58% (95% CI: -20% to 90%) by RT-PCR. However, VEs by IC and RT-PCR appeared almost similar in higher disease severity: 81.5% (95% CI: 40-94%) by IC and 72% (95% CI: 7-92%) by RT-PCR at severity ≥7, while 97.5% (95% CI: 77-99.7%) by IC and 92% (95% CI: 58-98%) by RT-PCR at severity ≥11. We showed that RV-vaccinated children had 80% [OR = 0.192 (95% CI: 0.052-0.709) less chance to be detected by IC. CONCLUSION: Although the sensitivity and specificity of IC differ by brand type, generally, IC is not as sensitive as RT-PCR. Despite the VEs remain higher by IC, it looks comparable with that of RT-PCR in severe cases implying that VEs evaluated by IC against severe illness remain useful for VE-monitoring.

Correction to: A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

In the original publication of article [1], '20 × 101 copies', which is in the sentence 'As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 101 copies in 2 µL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)' changes to '2.0 × 101 copies' in results section. The publisher apologizes to the readers and authors for the inconvenience.

A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

BACKGROUND: JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS: A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS: The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS: The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.

Replication-incompetent rabies virus vector harboring glycoprotein gene of lymphocytic choriomeningitis virus (LCMV) protects mice from LCMV challenge.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. CONCLUSIONS/SIGNIFICANCE: These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV.

A Single Vaccination of Nonhuman Primates with Highly Attenuated Smallpox Vaccine, LC16m8, Provides Long-term Protection against Monkeypox.

Monkeypox virus (MPXV) causes human monkeypox (human MPX), which is a similar disease to smallpox in humans. A previous study showed that a single vaccination of monkeys with LC16m8, a highly attenuated smallpox vaccine, protected them from MPX from 4-5 weeks post-vaccination. In this study, we evaluated the long-term efficacy of a single vaccination with LC16m8 in a nonhuman primate model of MPXV infection. The monkeys were inoculated with either LC16m8, Lister (parental strain of LC16m8), or a mock-up vaccine, and then challenged with MPXV via a subcutaneous route, at 6 and 12 months after vaccination, which we compared with either Lister or the mock-up vaccination. The LC16m8 monkeys exhibited almost no MPX-associated symptoms, whereas most of the naïve monkeys died. LC16m8 generated the protective memory immune response against MPXV, as suggested by the immediate viremia reduction and the response of the IgG antibody. The results demonstrated that the vaccination of monkeys with a single dose of LC16m8 provided durable protection against MPXV for longer than one year after immunization. The results suggest that the vaccination of humans with LC16m8 could induce long-term protection against MPXV infection.

Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines.

BACKGROUND: Ebolaviruses induce lethal viral hemorrhagic fevers (VHFs) in humans and non-human primates, with the exceptions of Reston virus (RESTV), which is not pathogenic for humans. In human VHF cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the IgG responses to nucleoprotein (NP) and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein (GP1,2) are the major indicator of protective immunity. On the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against NP and GP1,2 in RESTV-infected cynomolgus macaques is still unclear. In this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the RESTV epizootic in 1996 in the Philippines to expand our knowledge on the immune responses in naturally RESTV-infected non-human primates. RESULTS: The antibody responses were analyzed using IgG-ELISA, an indirect immunofluorescent antibody assay (IFA), and a pseudotyped VSV-based neutralizing (NT) assay. Antigen-capture (Ag)-ELISA was also performed to detect viral antigens in the serum specimens. We found that the anti-GP1,2 responses, but not the anti-NP responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the RESTV-infected cynomolgus macaques. Additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as IFNγ, IL8, IL-12, and MIP1α, in the convalescent phase sera. CONCLUSIONS: These results imply that both the antibody response to GP1,2 and the proinflammatory innate responses play significant roles in the recovery from RESTV infection in cynomolgus macaques.

A seroepidemiologic study of Reston ebolavirus in swine in the Philippines.

BACKGROUND: Ebola viruses cause viral hemorrhagic fever in humans and non-human primates and are endemic in Africa. Reston ebolavirus (REBOV) has caused several epizootics in cynomolgus monkeys (Macaca fascicularis) but is not associated with any human disease. In late 2008, REBOV infections were identified in swine for the first time in the Philippines. METHODS: A total of 215 swine sera collected at two REBOV-affected farms in 2008, in Pangasinan and Bulacan, were tested for the presence of REBOV-specific antibodies using multiple serodiagnosis systems. A total of 98 swine sera collected in a non-epizootic region, Tarlac, were also tested to clarify the prevalence of REBOV infection in the general swine population in the Philippines. RESULTS: Some 70 % of swine sera at the affected farms were positive for REBOV antibodies in the multiple serodiagnosis systems. On the other hand, none of the swine sera collected in Tarlac showed positive reactions in any of the diagnosis systems. CONCLUSIONS: The high prevalence of REBOV infection in swine in the affected farms in 2008 suggests that swine is susceptible for REBOV infection. The multiple serological assays used in the study are thought to be useful for future surveillance of REOBV infection in swine in the Philippines.

Expression of herpes simplex virus type 1 recombinant thymidine kinase and its application to a rapid antiviral sensitivity assay.

Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV(r)) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10 days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV(r) HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC(50)s) and inhibitory effects of these compounds on the replication of TAR among ACV(s) and ACV(r) HSV-1 clones. These results indicate that the EC(50)s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC(50) for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesion's samples or from virus isolates.

Virulence and pathophysiology of the Congo Basin and West African strains of monkeypox virus in non-human primates.

Monkeypox virus is divided into Congo Basin and West African strains. The virulence and pathophysiology of two strains, Zr-599 (a Congo Basin monkeypox virus) and Liberia (a West African monkeypox virus), were evaluated in non-human primates. Four monkeys were infected by the subcutaneous (SC) and two by the intranasal (IN) inoculation routes for Zr-599 and Liberia at a dose of 10(6) p.f.u. One monkey in the Liberia/SC group was demonstrated to be co-infected with Gram-positive cocci and was excluded from analyses. Infections in three of the four Zr-599/SC monkeys and in one of the three Liberia/SC monkeys were fatal. Virus genome levels in blood in the Zr-599/SC monkeys were approximately 10 times higher than those in the Liberia/SC monkeys. Zr-599 affected respiratory, genito-urinary and gastrointestinal tract organs more severely than Liberia. Zr-599 was more virulent than Liberia and one of the factors might be the difference in organ tropism.

Loop-mediated isothermal amplification-based diagnostic assay for monkeypox virus infections.

Monkeypox virus (MPXV) causes a smallpox-like disease in non-human primates and humans. This infection is endemic to central and western Africa. MPXV is divided into two genetically different groups, Congo Basin and West African MPXV, with the former being the more virulent. A real-time quantitative MPXV genome amplification system was developed for the diagnosis of MPXV infections using loop-mediated isothermal amplification (LAMP) technology. Primers used for genome amplification of Congo Basin (C-LAMP), West African (W-LAMP), and both Congo Basin and West African (COM-LAMP) MPXV by LAMP were designed according to the nucleotide sequences of the Congo Basin-specific D14L gene, the West African-specific partial ATI gene, and the partial ATI gene that is shared by both groups, respectively. The sensitivity and specificity of the LAMP were evaluated with nested PCR using peripheral blood and throat swab specimens collected from Congo Basin MPXV or West African MPXV-infected monkeys. The sensitivity and specificity of COM-LAMP, C-LAMP, and W-LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively. The viremia level determined by LAMP assays increased with increases in the severity of the monkeypox-associated symptoms. The newly developed LAMP assay was confirmed to be a rapid, quantifiable, and highly sensitive and specific system effective in the diagnosis of MPXV infections. The LAMP assays made it possible to discriminate between Congo Basin and West African MPXV. The LAMP developed in this study is useful not only for diagnosis of but also for the assessment of MPXV infections.

Diagnosis and assessment of monkeypox virus (MPXV) infection by quantitative PCR assay: differentiation of Congo Basin and West African MPXV strains.

Human monkeypox, an infectious disease caused by monkeypox virus (MPXV), is endemic to western and central Africa. A LightCycler quantitative PCR (LC-qPCR) system was developed for the diagnosis of this disease, targeting the A-type inclusion body gene (ATI gene) of MPXV. One naive monkey was infected with MPXV Zr-599 (Congo Basin strain) and one with MPXV Liberia (West African strain). Another three monkeys were immunized with smallpox vaccine on 0, 3, or 7 days, respectively, before infection with MPXV Zr-599. Peripheral blood cell (PBC) and throat swab (TS) specimens were serially collected. The LC-qPCR was validated for the diagnosis of monkeypox using virus isolation. Sequencing of the partial ATI gene revealed the insertion of a unique 453-nucleotide residue in the West African strains but not in the Congo Basin strains. Specific reverse primers for Congo Basin and West African strains were designed based on the unique sequence insertion. The LC-qPCR detected the MPXV genome, but not those of the other orthopoxviruses tested nor the varicella-zoster virus. Both the sensitivity and specificity of the LC-qPCR were over 90% in comparison to virus isolation when TS specimens were tested. Fourteen of the 15 virus isolation-positive PBC specimens showed positive reactions in the assay. Further, most PBC specimens collected from symptomatic monkeys in the later stage of illness showed positive reactions in the assay but negative reaction in virus isolation. It was possible to differentiate between these two groups with the LC-qPCR. Thus, the newly developed LC-qPCR is a useful and reliable diagnostic tool for MPXV infection.