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Cyprinid herpesvirus 3 (CyHV-3) is the archetypal fish alloherpesvirus and the etiologic agent of a lethal disease in common and koi carp. To date, the genome sequences of only four CyHV-3 isolates have been published, but no comparisons of the biologic properties of these strains have been reported. We have sequenced the genomes of a further seven strains from various geographical sources, and have compared their growth in vitro and virulence in vivo. The major findings were: (i) the existence of the two genetic lineages previously described as European and Asian was confirmed, but inconsistencies between the geographic origin and genotype of some strains were revealed; (ii) potential inter-lineage recombination was detected in one strain, which also suggested the existence of a third, as yet unidentified lineage; (iii) analysis of genetic disruptions led to the identification of non-essential genes and their potential role in virulence; (iv) comparison of the in vitro and in vivo properties of strains belonging to the two lineages revealed that inter-lineage polymorphisms do not contribute to the differences in viral fitness observed; and (v) a negative correlation was observed among strains between viral growth in vitro and virulence in vivo. This study illustrates the importance of coupling genomic and biologic comparisons of viral strains in order to enhance understanding of viral evolution and pathogenesis.

The amino acid at position 55 of the E2 glycoprotein (E2 55 ) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E2 55 ‫؍‬ histidine) differs only at this position from virus strain 633 (E2 55 ‫؍‬ glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes (ϳ50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E2 55 impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence.Sindbis virus (SV), the prototype member of the genus Alphavirus in the family Togaviridae, causes age-dependent mortality in mice (23). In newborn mice, the AR339 strain of SV causes acute fatal encephalomyeliitis while in older mice the disease is mild and nonfatal. A neuroadapted strain of SV isolated after serial intracerebral passages of SV causes high mortality in older mice associated with severe encephalomyelitis resulting in kyphoscoliosis and hindlimb paralysis (24). Fatal infection correlates with the induction of neuronal death, the primary target cells of SV infection in the nervous system (34).SV is a plus-strand RNA virus with a cell-derived lipid bilayer membrane containing heterodimers of the two viral envelope glycoproteins, E1 and E2 (51). Entry of SV into host cells involves association of the virus with the cell surface, interaction with specific receptors, clathrin-mediated endocytosis, and acid-induced fusion of SV with the endosomal membrane to release the nucleocapsid into the cytoplasm (29). Coordinated association and dissociation of the E1 and E2 glycoproteins during interaction with the cellular me...

Viral DNA and RNA polymerases are enzymes, which are responsible for copying the genetic materials of viruses and are therefore central components in the life cycles of viruses. The polymerases are essentially required for the replication of viruses. The reverse transcriptase (RT) of the retroviruses and the hepadnaviruses is the sole viral enzyme required for the synthesis of DNA from viral RNA. Viral polymerases are therefore an extremely favorable target for the development of antiviral therapy. The success of anti-HIV-1 therapy using inhibitors specifically targeting HIV RT suggests that other viral polymerases can be the valid molecular targets for the design of antiviral drugs. Intensive structural and functional studies of viral polymerases have been conducted and have opened new avenues for the development of more effective antiviral therapy. This review summarizes the insights gained from recent structural and functional studies of antiviral agents, which target viral polymerases. The primary focus will be on hepatitis C virus (HCV), herpesviruses, HIV, hepatitis B virus (HBV) and influenza virus.

e Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3= untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n ‫؍‬ 220) and individuals not suspected of dengue virus infection (n ‫؍‬ 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. Dengue virus (DENV) is a mosquito-borne human pathogen that belongs to the genus Flavivirus in the family Flaviviridae (1). DENV is an enveloped virus with a single-stranded, positivesense RNA genome approximately 10.7 kb in length (2). The genomic RNA includes 5= and 3= untranslated regions (UTRs) and a single open reading frame that encodes a single polyprotein that is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3). DENV is comprised of four distinct serotypes (DENV-1, -2, -3, and -4), and all four serotypes are currently circulating in the Pacific Islands, Americas, Asia, and Africa. In addition to this, distinct genotypes have been identified within each serotype (4, 5), highlighting the extensive genetic variability of the DENV serotypes.DENV infection is considered a major public health problem in developing tropical countries where the virus is endemic, and it is continuously spreading to new geographical areas around the world (6, 7). Moreover, frequent international travel to regions where dengue is endemic or epidemic increases the risk of DENV infection for travelers (8,9), and travel contributes to the escalating numbers of imported dengue cases in temperate regions. More than 2.5 billion people ...