Yellow fever, a mosquito-borne disease, is an important viral hemorrhagic fever in Africa and South America where it is endemic. Detection of yellow fever virus (YFV) in Africa remains a challenge due to a lack of highly specific tests. The aim of this study was to develop and optimize a rapid detection reverse transcription loop-mediated isothermal amplification (RT-LAMP) for YFV. The RT-LAMP was done isothermally at 62°C using a real-time turbidimeter that allowed detection within 1 hr. Specificity of the RT-LAMP was determined using RNA from flaviviruses and other related viruses where only YFV RNA was detected: West Nile virus, dengue viruses, Japanese encephalitis virus, Rift Valley fever virus, and chikungunya virus. In addition, equal sensitivity was also observed when the RT-LAMP and the real-time RT-PCR were compared using YFV-spiked human serum samples with a detection limit of 0.29 PFU/ml. Two Kenyan YFV wild strains showed an equal detection limit as the vaccine strain 17D in this study. The RT-LAMP reduced the time of reaction from 3hours to 1hour and increased sensitivity tenfold compared to RT-PCR. Therefore, this test offers a simple, rapid and reliable diagnostic tool for yellow fever when there are outbreaks of acute hemorrhagic fever in Kenya and other African countries.

The National Notifiable Diseases Surveillance System (NNDSS) received notification of 9,291 cases of disease transmitted by mosquitoes during the 2010-11 season (1 July 2010 to 30 June 2011). The alphaviruses Barmah Forest virus and Ross River virus accounted for 7,515 (81%) of these. There were 133 notifications of dengue virus infection acquired in Australia and 1,133 cases that were acquired overseas, while for 10 cases, the place of acquisition was unknown. The number of overseas acquired cases of dengue continues to rise each year, and these are most frequently acquired in Indonesia. Sentinel chicken, mosquito surveillance, viral detection in mosquitoes and climate modelling are used to provide early warning of arboviral disease activity in Australia. In early 2011, sentinel chickens in south eastern Australia widely seroconverted to flaviviruses. In 2010-11, there were 16 confirmed human cases of Murray Valley encephalitis acquired in Australia. There was one human case of Kunjin virus infection. There were 7 notifications of locally-acquired malaria in Australia and 407 notifications of overseas-acquired malaria during the 2010-11 season. Commun Dis Intell 2013;37:E1-E20.

[This corrects the article on p. e52909 in vol. 8.].

PURPOSE:

Acanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as "Acanthamoeba keratitis" (AK) and, more rarely, an infection of the central nervous system called "granulomatous amoebic encephalitis" (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. Moxifloxacin, a fourth generation fluoroquinolone, has been used with other drugs to treat GAE, but its efficacy as a treatment for AK is not known. Voriconazole has been used to treat AK; however, its cysticidal efficacy is not known. Both drugs are commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba compared to two reference drugs (chlorhexidine and amphotericin B) which are currently used to treat AK and GAE.

METHODS:

The sensitivity of two clinical and one type strain of Acanthamoeba to the commercial concentrations of the four drugs was evaluated with a colorimetric assay. Mature cysts were incubated with voriconazole to determine their sensitivity to this drug. The effects on cell proliferation and cell toxicity were determined using standard procedures with commercial kits.

RESULTS:

The four compounds were active against the Acanthamoeba strains in this study. Although it prevented encystation, moxifloxacin's amoebicidal activity was low. Voriconazole activity was greater than that of the other drugs, even at a concentration lower than in commercial eye drops. It was effective against cysts and decreased cell proliferation, with low cellular cytotoxicity.

CONCLUSION:

Voriconazole could be used against AK as a first-line treatment or in combination. Moxifloxacin is an interesting adjuvant to consider as it is effectively prevents encystation of the amoeba which often complicates infection resolution. In addition, moxifloxacin is effective in preventing secondary bacterial infections.

The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.

Human herpesvirus-6B (HHV-6B) is a T lymphotropic β-herpesvirus that is clearly distinct from human herpesvirus-6A (HHV-6A) according to molecular biological features. The International Committee on Taxonomy of Viruses recently classified HHV-6B as a separate species. The primary HHV-6B infection causes exanthem subitum and is sometimes associated with severe encephalopathy. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection. HHV-6B reactivation causes encephalitis in immunosuppressed patients. The cellular receptor for HHV-6A entry was identified as human CD46, but the receptor for HHV-6B has not been clear. Here we found that CD134, a member of the TNF receptor superfamily, functions as a specific entry receptor for HHV-6B. A T-cell line that is normally nonpermissive for HHV-6B infection became highly susceptible to infection when CD134 was overexpressed. CD134 was down-regulated in HHV-6B-infected T cells. Soluble CD134 interacted with the HHV-6B glycoprotein complex that serves as a viral ligand for cellular receptor, which inhibited HHV-6B but not HHV-6A infection in target cells. The identification of CD134 as an HHV-6B specific entry receptor provides important insight into understanding HHV-6B entry and its pathogenesis.

A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 10(2) copies and a linear dynamic range of 10(5) to 10(10) copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies.

Herpes simplex encephalitis classically involves the periventricular white matter in infants and the mesial temporal lobes, inferior frontal lobes, and insula in older children and adults. However, the increasing use of polymerase chain reaction to detect viral DNA in the cerebrospinal fluid has allowed the expansion of the spectrum of radiologic findings possibly associated with herpes simplex encephalitis. This study presents a rare case of a previously healthy infant with herpes simplex encephalitis with occipital involvement and permanent visual impairment. Possible pathogenic mechanisms are discussed.

Schmallenberg virus (SBV) is a recently discovered Bunyavirus associated mainly with abortions, stillbirths and malformations of the skeletal and central nervous system (CNS) in newborn ruminants. In this study, a detailed immunophenotyping of the inflammatory cells of the CNS of affected animals was carried out in order to increase our understanding of SBV pathogenesis. A total of 82 SBV-polymerase chain reaction (PCR) positive neonatal ruminants (46 sheep lambs, 34 calves and 2 goat kids) were investigated for the presence of inflammation in the brain and spinal cord. The study focused on 15 out of 82 animals (18.3%) showing inflammation in the CNS. All 15 neonates displayed lymphohistiocytic meningoencephalomyelitis affecting most frequently the mesencephalon and the parietal and temporal lobes. The majority of infiltrating cells were CD3-positive T cells, followed by CD79α-positive B cells and CD68-positive microglia/macrophages. Malformations like por- and hydranencephaly, frequently found in the temporal lobe, showed associated demyelination and axonal loss. SBV antigen was detected in 37 out of 82 (45.1%) neonatal brains by immunohistochemistry. In particular, SBV antigen was found in 93.3% (14 out of 15 ruminants) and 32.8% (22 out of 67 ruminants) of animals with and without encephalitis, respectively. Highest amounts of virus-protein expression levels were found in the temporal lobe. Our findings suggest that: (i) different brain regions display differential susceptibility to SBV infection; (ii) inflammatory cells in the CNS are found only in a minority of virus infected animals; (iii) malformations occur in association with and without inflammation in the CNS; and (iv) viral antigen is strongly associated with the presence of inflammation in naturally infected animals. Further studies are required to explore the cell tropism and pathogenesis of SBV infection in ruminants.