Comparative mutational analyses of influenza A viruses.

The error-prone RNA-dependent RNA polymerase (RdRP) and external selective pressures are the driving forces for RNA viral diversity. When confounded by selective pressures, it is difficult to assess if influenza A viruses (IAV) that have a wide host range possess comparable or distinct spontaneous mutational frequency in their RdRPs. We used in-depth bioinformatics analyses to assess the spontaneous mutational frequencies of two RdRPs derived from human seasonal (A/Wuhan/359/95; Wuhan) and H5N1 (A/Vietnam/1203/04; VN1203) viruses using the mini-genome system with a common firefly luciferase reporter serving as the template. High-fidelity reverse transcriptase was applied to generate high-quality mutational spectra which allowed us to assess and compare the mutational frequencies and mutable motifs along a target sequence of the two RdRPs of two different subtypes. We observed correlated mutational spectra (τ correlation P < 0.0001), comparable mutational frequencies (H3N2:5.8 ± 0.9; H5N1:6.0 ± 0.5), and discovered a highly mutable motif "(A)AAG" for both Wuhan and VN1203 RdRPs. Results were then confirmed with two recombinant A/Puerto Rico/8/34 (PR8) viruses that possess RdRP derived from Wuhan or VN1203 (RG-PR8×Wuhan(PB2, PB1, PA, NP) and RG-PR8×VN1203(PB2, PB1, PA, NP)). Applying novel bioinformatics analysis on influenza mutational spectra, we provide a platform for a comprehensive analysis of the spontaneous mutation spectra for an RNA virus.

MERS coronavirus in dromedary camel herd, Saudi Arabia.

A prospective study of a dromedary camel herd during the 2013-14 calving season showed Middle East respiratory syndrome coronavirus infection of calves and adults. Virus was isolated from the nose and feces but more frequently from the nose. Preexisting neutralizing antibody did not appear to protect against infection.

Chemokine up-regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells.

Lymphopenia and increasing viral load in the first 10 days of severe acute respiratory syndrome (SARS) suggested immune evasion by SARS-coronavirus (CoV). In this study, we focused on dendritic cells (DCs) which play important roles in linking the innate and adaptive immunity. SARS-CoV was shown to infect both immature and mature human monocyte-derived DCs by electron microscopy and immunofluorescence. The detection of negative strands of SARS-CoV RNA in DCs suggested viral replication. However, no increase in viral RNA was observed. Using cytopathic assays, no increase in virus titer was detected in infected DCs and cell-culture supernatant, confirming that virus replication was incomplete. No induction of apoptosis or maturation was detected in SARS-CoV-infected DCs. The SARS-CoV-infected DCs showed low expression of antiviral cytokines (interferon alpha [IFN-alpha], IFN-beta, IFN-gamma, and interleukin 12p40 [IL-12p40]), moderate up-regulation of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and IL-6) but significant up-regulation of inflammatory chemokines (macrophage inflammatory protein 1alpha [MIP-1alpha], regulated on activation normal T cell expressed and secreted [RANTES]), interferon-inducible protein of 10 kDa [IP-10], and monocyte chemoattractant protein 1 [MCP-1]). The lack of antiviral cytokine response against a background of intense chemokine up-regulation could represent a mechanism of immune evasion by SARS-CoV.

A prospective case control study of the association of Gianotti-Crosti syndrome with human herpesvirus 6 and human herpesvirus 7 infections.

Gianotti-Crosti syndrome (GCS) is known to be associated with hepatitis B and Epstein-Barr virus (EBV) infections. Apart from a single case report based on serology alone, there are no published data on an association between GCS and human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) primary infections or reactivations. Our aim was to investigate the association between GCS and HHV-6 and HHV-7 infections. Ten patients diagnosed with GCS at a primary care practice over an 18-month period were recruited. Controls were age- and sex-matched patients with unrelated symptoms requiring venepuncture for other indications. Blood specimens were collected from patients and controls at presentation, and from patients 4 weeks later. Virologic evidence of HHV-6 and HHV-7 infection was sought in peripheral blood leukocytes and plasma using polymerase chain reaction (PCR) for viral DNA, reverse transcriptase polymerase chain reaction (RT-PCR) for HHV-6 U91 mRNA transcripts, and serology. Serology for EBV and hepatitis B virus was done. In contrast to the 10 controls, 2 patients (both infants) with clinically diagnosed GCS had evidence of active HHV-6 infection. This was demonstrated by detection of viral DNA in the absence of antibody in the acute plasma specimens and HHV-6 DNA viral loads of more than 5.3 log10 genome copies/5 microl in the whole blood specimens, a profile previously shown to be diagnostic of recent primary HHV-6 infection. None of the patients had evidence of recent EBV or hepatitis B infection. We conclude that primary HHV-6 infection may be associated with GCS in some infants.