Expanding the range of the respiratory infectome in Australian feedlot cattle with and without respiratory disease using metatranscriptomics.

BACKGROUND: Bovine respiratory disease (BRD) is one of the most common diseases in intensively managed cattle, often resulting in high morbidity and mortality. Although several pathogens have been isolated and extensively studied, the complete infectome of the respiratory complex consists of a more extensive range unrecognised species. Here, we used total RNA sequencing (i.e., metatranscriptomics) of nasal and nasopharyngeal swabs collected from animals with and without BRD from two cattle feedlots in Australia. RESULTS: A high abundance of bovine nidovirus, influenza D, bovine rhinitis A and bovine coronavirus was found in the samples. Additionally, we obtained the complete or near-complete genome of bovine rhinitis B, enterovirus E1, bovine viral diarrhea virus (sub-genotypes 1a and 1c) and bovine respiratory syncytial virus, and partial sequences of other viruses. A new species of paramyxovirus was also identified. Overall, the most abundant RNA virus, was the bovine nidovirus. Characterisation of bacterial species from the transcriptome revealed a high abundance and diversity of Mollicutes in BRD cases and unaffected control animals. Of the non-Mollicutes species, Histophilus somni was detected, whereas there was a low abundance of Mannheimia haemolytica. CONCLUSION: This study highlights the use of untargeted sequencing approaches to study the unrecognised range of microorganisms present in healthy or diseased animals and the need to study previously uncultured viral species that may have an important role in cattle respiratory disease. Video Abstract.

Multiplexed serotype-specific real-time polymerase chain reaction assays - A valuable tool to support large-scale surveillance for bluetongue virus infection.

In the last decade, real-time polymerase chain reaction (PCR) has been increasingly adopted for bluetongue diagnosis with both broadly reactive and serotype-specific assays widely used. The use of these assays and nucleic acid sequencing technologies have enhanced bluetongue virus detection, resulting in the identification of a number of new serotypes. As a result, 27 different serotypes are officially recognised, and at least three more are proposed. Rapid identification of the virus serotype is essential for matching of antigens used in vaccines and to undertake surveillance and epidemiological studies to assist risk management. However, it is not uncommon for multiple serotypes to circulate in a region either concurrently or in successive years. It is therefore necessary to have a large suite of assays available to ensure that the full spectrum of viruses is detected. Nevertheless, covering a large range of virus serotypes is demanding from both a time and resource perspective. To overcome these challenges, real-time PCR assays were optimised to match local virus strains and then combined in a panel of quadriplex assays, resulting in three assays to detect 12 serotypes directly from blood samples from cattle and sheep. These multiplex assays have been used extensively for bluetongue surveillance in both sentinel animals and opportunistically collected samples. A protocol to adapt these assays to capture variations in local strains of bluetongue virus and to expand the panel is described. Collectively, these assays provide powerful tools for surveillance and the rapid identification of bluetongue virus serotypes directly from animal blood samples.

Progress in public health risk communication in China: lessons learned from SARS to H7N9.

BACKGROUND: Following the SARS outbreak, the World Health Organization revised the International Health Regulations to include risk communication as one of the core capacity areas. In 2006, the U.S. Centers for Disease Control and Prevention's Global Disease Detection [GDD] program began collaborating with China to enhance China's risk communication capacity to address gaps in the SARS communication response. This article describes tangible improvements in China's public health emergency risk communication capacity between the SARS and H7N9 outbreaks; documents U.S. CDC GDD cooperative technical assistance during 2006-2017; and shares lessons learnt to benefit other countries and contribute to enhance global health security. METHOD: A questionnaire based on the WHO Joint External Evaluation tool [Risk Communication section] was developed. A key communications official from the China National Health Commission [NHC] completed the questionnaire retrospectively to reflect China's capacity to manage communication response before, during and after the outbreaks of SARS in 2003, influenza H1N1 in 2009, and influenza H7N9 in 2013. A literature search was also conducted in English and Chinese to further substantiate the results of the questionnaire completed by NHC. RESULTS: China demonstrated significantly improved risk communication capacities of pre-event, during event and post event responses to H7N9 when compared to the SARS response. China NHC improved its response through preparedness, availability of dedicated staff and resources for risk communication, internal clearance mechanisms, standard operating procedures with national response parties external to NHC, rumor management, communication with international agencies and consistent messaging with healthcare and private sectors. Correspondingly, the perceived level of trust that the public had in the NHC following outbreaks rose between the SARS and H7N9 response. CONCLUSION: Risk communication capacities in China have increased during the ten years between the SARS outbreak of 2003 and the H7N9 outbreak of 2013. Long-term risk communication capacity building efforts in bilateral collaborations are uncommon. The U.S. CDC GDD project was one of the first such collaborations worldwide. The lessons learned from this project may benefit lower and middle-income countries as they build their national emergency risk communication capacity.

Identification of a novel nidovirus as a potential cause of large scale mortalities in the endangered Bellinger River snapping turtle (Myuchelys georgesi).

In mid-February 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, Myuchelys georgesi, in a coastal river in New South Wales, Australia. Mortalities continued for approximately 7 weeks and affected mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. At necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. Histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. A virus was isolated in cell culture from a range of tissues. Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. In contrast, in the affected turtles, the most significant pathological changes were in the kidneys. Real time PCR assays developed to detect this virus demonstrated very high virus loads in affected tissues. In situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. Collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. Bellinger River Virus is the name proposed for this new virus.

Perceptions on the risk communication strategy during the 2013 avian influenza A/H7N9 outbreak in humans in China: a focus group study.

OBJECTIVE: To identify the general public's perceptions of the overall risk communication strategy carried out by Chinese public health agencies during the first wave of avian influenza A(H7N9) outbreak in humans in 2013. METHODS: Participants were recruited from communities in Beijing, Lanzhou and Hangzhou, China in May and June 2013 by convenience sampling. Demographics and other relevant information were collected using a self-administered questionnaire. Focus group interviews were conducted using a set of nine pre-developed questions and a tested moderator guide. The interviews were audio recorded and were transcribed verbatim. The constant comparative method was used to identify trends and themes. RESULTS: A total of nine focus group interviews, with 94 participants recruited from nine communities, were conducted. Most participants received H7N9 information via television and the Internet. Most the participants appreciated the transparency and timeliness of the information released by the government. They expressed a sense of trust in the recommended public health advice and followed most of them. The participants suggested that the government release more information about clinical treatment outcomes, have more specific health recommendations that are practical to their settings and expand the use of new media channels for risk communication. CONCLUSION: The public perceived the overall risk communication strategy by the Chinese public health agencies as effective, though the moderator had a governmental agency title that might have biased the results. There is a need to expand the use of social media for risk communication in the future.

Assessing knowledge and application of emergency risk communication principles among public health workers in China.

OBJECTIVE: Since 2003, the Chinese National Health and Family Planning Commission (formerly the Ministry of Health) has implemented changes to more effectively communicate risk during public health emergencies. In spite of ongoing improvements, provincial and sub-provincial leaders face barriers, such as established modes of operation, lack of training, shortage of trained risk communicators, and limited understanding and willingness of recipients to mitigate risks. METHODS: We assessed the current status of and barriers to risk communication knowledge and practice among public health practitioners in China. We designed the survey questionnaire to capture information related to the risk communication core capacities required by international health regulations and common risk communication principles. RESULTS: Our findings showed that risk communication training has successfully developed an awareness of risk communication principles and the ability to implement those principles in practice in China. CONCLUSIONS: Future efforts should focus on areas such as a dedicated risk communication workforce, requirements that public health agencies develop a risk communication plan, and additional training for public health practitioners and their partners. It is critical that the infectious diseases prevention and control law be amended to grant provincial and local public health agencies more autonomy to release information.

Community-level text messaging for 2009 H1N1 prevention in China.

BACKGROUND: Although patients worldwide increasingly are using mobile phone text messaging (SMS) for clinical care, quality data are sparse on the community-level effectiveness of SMS to prevent and control disease. PURPOSE: To determine SMS effectiveness in improving 2009 H1N1 knowledge, attitudes, behaviors, and self-reported outcomes and to assess community SMS acceptability. METHODS: A program evaluation of Shanghai, China's SMS system using a single-blinded, randomized-controlled method was conducted in 2010 and results were analyzed in 2010-2011. Randomly selected community residents who agreed to participate were assigned to receive 3 weeks of either 2009 H1N1 prevention and control or tobacco-cessation messages. Assessments were made of 2009 H1N1 knowledge, attitudes, behaviors, and self-reported influenza-like illness before and after sending messages to participants. Acceptability of SMS also was assessed. RESULTS: Of 1992 respondents, those receiving 2009 H1N1 messages had higher scores measuring 2009 H1N1 knowledge (4.2% higher) and desired attitudes (9.4% higher) (p<0.001); 1.77 times greater odds of new 2009 H1N1 vaccination (p<0.001); and 0.12 times smaller odds of reporting influenza-like illness (p<0.001) than those receiving tobacco messages. More than 95% of participants found the SMS program useful and trustworthy; nearly 90% would use it again. CONCLUSIONS: SMS can improve self-reported uptake of short-term behaviors, such as vaccination, that can result in long-term prevention and control of disease. SMS can improve knowledge and influence attitudes about infection prevention and control and self-reported health outcomes. In Shanghai, health-based SMS is acceptable to users.

Characterization of virulent West Nile virus Kunjin strain, Australia, 2011.

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.

Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia.

In March 2010, an outbreak of low pathogenicity avian influenza A (H10N7) occurred on a chicken farm in Australia. After processing clinically normal birds from the farm, 7 abattoir workers reported conjunctivitis and minor upper respiratory tract symptoms. Influenza virus A subtype H10 infection was detected in 2 workers.

Experimental infections of the porcine foetus with Bungowannah virus, a novel pestivirus.

In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn pigs and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to amplify any infectious agents present in field material to aid the detection and identification of the causative agent of the porcine myocarditis syndrome (PMC). Foetuses were directly inoculated in utero with tissue extracts from field cases of PMC at 56-60, 70-84 or 85-94 days of gestation and euthanased 7-28 days later. The IgG concentration in foetal sera/body fluids was measured, hearts were examined by light microscopy and selected hearts were examined by electron microscopy. An infectious agent was detected in tissues from cases of PMC and its identification as the novel pestivirus Bungowannah virus has recently been reported (Kirkland et al., 2007). Sow sera, foetal tissues and foetal sera/body fluids were tested for Bungowannah virus RNA by qRT-PCR and antibody by peroxidase-linked assay. Bungowannah virus was detected in numerous organs of the porcine foetus. Following direct foetal exposure it is probable that this virus spreads by direct intra-uterine transmission to adjacent foetuses and by trans-uterine transmission to the dam. Data were obtained for both the replication of the virus in the porcine foetus and the humoral immune response in the foetus and sow.

Field and laboratory evidence that Bungowannah virus, a recently recognised pestivirus, is the causative agent of the porcine myocarditis syndrome (PMC).

In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn piglets and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to review existing data and to undertake further investigations of specimens from naturally infected pigs to provide evidence to support the hypothesis that Bungowannah virus, a recently recognised pestivirus, causes the porcine myocarditis syndrome (PMC). Sera collected from gilts and sows from affected and unaffected units were tested for Bungowannah virus antibody by a peroxidase-linked assay and Bungowannah virus RNA by qRT-PCR in selected cases. Stillborn piglets from affected and an unaffected unit were also tested for Bungowannah virus antibody and RNA. Body fluid IgG levels and the incidence of myocardial lesions in these stillborn piglets are summarised. Tissue sections from stillborn piglets with myocarditis/myonecrosis were examined for Bungowannah virus RNA by in situ hybridisation. A clear temporal association between the occurrence of PMC on a unit or module and exposure to Bungowannah virus was identified by serological tests in both breeding aged animals and stillborn pigs. In addition, at the individual animal level on affected units, Bungowannah virus RNA was detected in stillborn piglets in large amounts by qRT-PCR and in association with myocardial lesions by in situ hybridisation. The examination of field material from cases of PMC by serology, qRT-PCR and in situ hybridisation provides strong indirect evidence that Bungowannah virus is the causative agent for PMC.

Immune cell transcriptome datasets reveal novel leukocyte subset-specific genes and genes associated with allergic processes.

BACKGROUND: The precise function of various resting and activated leukocyte subsets remains unclear. For instance, mast cells, basophils, and eosinophils play important roles in allergic inflammation but also participate in other immunologic responses. One strategy to understand leukocyte subset function is to define the expression and function of subset-restricted molecules. OBJECTIVE: To use a microarray dataset and bioinformatics strategies to identify novel leukocyte markers as well as genes associated with allergic or innate responses. METHODS: By using Affymetrix microarrays, we generated an immune transcriptome dataset composed of gene profiles from all of the major leukocyte subsets, including rare enigmatic subsets such as mast cells, basophils, and plasma cells. We also assessed whether analysis of genes expressed commonly by certain groups of leukocytes, such as allergic leukocytes, might identify genes associated with particular responses. RESULTS: Transcripts highly restricted to a single leukocyte subset were readily identified (>2000 subset-specific transcripts), many of which have not been associated previously with leukocyte functions. Transcripts expressed exclusively by allergy-related leukocytes revealed well known as well as novel molecules, many of which presumably contribute to allergic responses. Likewise, Nearest Neighbor Analysis of genes coexpressed with Toll-like receptors identified genes of potential relevance for innate immunity. CONCLUSION: Gene profiles from all of the major human leukocyte subsets provide a powerful means to identify genes associated with single leukocyte subsets, or different types of immune response. CLINICAL IMPLICATIONS: A comprehensive dataset of gene expression profiles of human leukocytes should provide new targets or biomarkers for human inflammatory diseases.

The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation.

The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma.