Paul F. Horwood

Repertoire of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection.

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illuminas ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.

Outbreak of Chikungunya Virus Infection, Vanimo, Papua New Guinea

In June 2012, health authorities in Papua New Guinea detected an increase in febrile illnesses in Vanimo. Chikungunya virus of the Eastern/Central/Southern African genotype harboring the E1:A226V mutation was identified. This ongoing outbreak has spread to ≥8 other provinces and has had a harmful effect on public health.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

Preparedness for threat of Chikungunya in the Pacific

Chikungunya virus (CHIKV) caused significant outbreaks of illness during 2005–2007 in the Indian Ocean region. Chikungunya outbreaks have also occurred in the Pacific region, including in Papua New Guinea in 2012; New Caledonia in April 2013; and Yap State, Federated States of Micronesia, in August 2013. CHIKV is a threat in the Pacific, and the risk for further spread is high, given several similarities between the Pacific and Indian Ocean chikungunya outbreaks. Island health care systems have difficulties coping with high caseloads, which highlights the need for early multidisciplinary preparedness. The Pacific Public Health Surveillance Network has developed several strategies focusing on surveillance, case management, vector control, laboratory confirmation, and communication. The management of this CHIKV threat will likely have broad implications for global public health.

Characterization of microRNAs encoded by the bovine herpesvirus 1 genome

Bovine herpesvirus 1 (BoHV-1) is a ubiquitous and important pathogen of cattle worldwide. This study reports the identification of 10 microRNA (miRNA) genes, Bhv1-mir-B1-Bhv1-mir-B10, encoded by the BoHV-1 genome that were processed into 12 detectable mature miRNAs as determined by ultra-high throughput sequencing bioinformatics analyses of small RNA libraries and expression studies. We found that four of the miRNA genes were present as two copies in the BoHV-1 genome, resulting in a total of 14 miRNA encoding loci. Unique features of the BoHV-1 miRNAs include evidence of bidirectional transcription and a close association of two miRNA genes with the origin of replication, including one miRNA that is encoded within the origin of replication. The miRNA gene Bhv1-mir-B5 was encoded on the opposite DNA strand to the latency associated transcript, potentially giving rise to antisense transcripts originating from this locus. The association of herpesvirus miRNAs with latency appears to be a common feature in the alphaherpesviruses. Analyses of the BoHV-5 genome for putative miRNA gene orthologues identified a high degree of evolutionary conservation for nine of the BoHV-1 miRNA genes. The possible roles for BoHV-1 miRNAs in the regulation of known BoHV-1 transcription units and the genetics of the BoHV-1 genotypes are also discussed.

Influenza antiviral resistance in the Asia-Pacific region during 2011

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Evaluation of Serological Diagnostic Tests for Typhoid Fever in Papua New Guinea Using a Composite Reference Standard

ABSTRACT Typhoid fever remains a major global health problem. A major impediment to improving outcomes is the lack of appropriate diagnostic tools, which have not significantly improved in low-income settings for 100 years. We evaluated two commercially available rapid diagnostic tests (Tubex and TyphiDot), a prototype (TyphiRapid TR-02), and the commonly used single-serum Widal test in a previously reported high-burden area of Papua New Guinea. Samples were collected from 530 outpatients with axillary temperatures of ≥37.5°C, and analysis was conducted on all malaria-negative samples (n = 500). A composite reference standard of blood culture and PCR was used, by which 47 participants (9.4%) were considered typhoid fever positive. The sensitivity and specificity of the Tubex (51.1% and 88.3%, respectively) and TyphiDot (70.0% and 80.1%, respectively) tests were not high enough to warrant their ongoing use in this setting; however, the sensitivity and specificity for the TR-02 prototype were promising (89.4% and 85.0%, respectively). An axillary temperature of ≥38.5°C correlated with typhoid fever (P = 0.014). With an appropriate diagnostic test, conducting typhoid fever diagnosis only on patients with high-grade fever could dramatically decrease the costs associated with diagnosis while having no detrimental impact on the ability to accurately diagnose the illness.

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

Background Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. Methodology/Principal Findings Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. Conclusions Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Multiplex real-time RT-PCR detection of three viruses associated with the bovine respiratory disease complex.

Bovine respiratory disease complex (BRDC) is considered the most significant illness associated with feedlot cattle in North America and possibly worldwide. BRDC is a multi-factorial disease with environmental conditions interacting with multiple viral and bacterial pathogens to produce severe respiratory illness. Bovine herpesvirus 1, bovine viral diarrhoea virus and bovine parainfluenza virus 3 are three of the major viruses associated with BRDC. In this study, a multiplex real-time RT-PCR using Taqman primers and probes was developed to detect simultaneously all three of these important BRDC viruses. The assay was optimised and validated using cell-culture infected material and bovine clinical samples from BRDC cases. The sensitivity of the assay was analysed by comparing the multiplex with the individual singleplex assays, which demonstrated that viral detection was not inhibited, by multiplexing the real-time RT-PCRs. This technique is the first reported real-time PCR method for BPIV-3 and the first multiplex designed to detect three BRDC viral pathogens.

Identification of Molecular Markers Associated with Alteration of Receptor-Binding Specificity in a Novel Genotype of Highly Pathogenic Avian Influenza A(H5N1) Viruses Detected in Cambodia in 2013

ABSTRACT Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.