In February and March of 2013, several patients in eastern China were hospitalized with severe lower respiratory tract infections, the causes of which were unknown. A previously unrecognized avian strain of influenza A virus (H7N9) was identified with these infections. By mid-April, 60 influenza H7N9 cases were confirmed in five Chinese provinces, spiking to 130 reported cases by May. The route of transmission was not clearly defined, but the flu strain was found in chickens, suggesting that close contact with poultry in live animal markets may be involved. June reports show that new cases of H7N9 influenza continue in ten provinces, with a relatively low mortality rate of ~20%. To date, no cases of H7N9 outside of China have been reported, and no confirmed human-to-human contact has been established. However, simultaneous infection of humans and animals with several influenza strains is being reported, raising concerns about viral genetic adaptations that could pave the way for emergence of an epidemic/pandemic strain with ability to spread quickly in human populations.
A well-known and highly infectious respiratory pathogen, influenza A is an enveloped, single-stranded RNA virus belonging to the Orthomyxoviridae family. Influenza pathogenesis is complex, involving multiple host species and reassortment of the eight viral genes that control receptor tropism and a range of other factors that dictate infection. Pandemic strains have peaked and receded over the centuries, the last in 2009 with a multiple reassortment strain called H1N1. With improvements in next-generation sequencing (NGS) technology, changes in viral genes can be tracked and traced in near real time. Genetic epidemiology provides great assistance to regional public health defenses in potential outbreak situations.
Illuminating the Situation
This spring, the Jiangsu Provincial Center for Disease Control and Prevention (CDC) wanted to use whole-genome sequence data to confirm H7N9 cases and to shed light on the possible transmission route of the virus. They chose the MiSeq sequencer as their preferred system for rapid and accurate sequencing. Working closely with Illumina’s team in China, a MiSeq instrument was placed in the CDC laboratory, and with comprehensive setup and support, it was quickly up and running. The CDC team took 8 samples, from both confirmed cases, unknown human samples, and from animal sources. They extracted total RNA, reverse transcribed it, and amplified cDNA with virus-specific primers. Using Nextera XT for sample preparation, they sequenced the samples on the MiSeq using 2 x 150 paired-end reads. Data were aligned against the published H7N9 sequence and variants were called using the MiSeq’s on-instrument software.
Positive identification of H7N9 was made. Within the tiny 13.5 Kb viral genome, the researchers found mutations that varied slightly in number or location from sample to sample, indicating that strains either had different origins or a fast mutation rate was occurring between transmission events. Phylogenetic analysis of the sequences and the differences between them determined the relatedness of the strains. Samples taken from chicken manure turned out to be closely related to at least one patient sample, confirming the likelihood of H7N9 transmission from chickens to humans, a hypothesis which can be strengthened with the inclusion of more samples.
In another experiment, full-genome sequencing of a virus isolated from a patient again confirmed infection with H7N9. This patient worked in a live-animal market, and environmental samples also turned up H7N9 that was 98.9% identical to the patient strain, in addition to various influenza strains typically found in chickens. Both H7N9 isolates contained variants that could lead to amino acid changes affecting the way the viral glycoprotein hemagglutinin (HA) binds to a cellular receptor called sialic acid present on upper respiratory tract cells of the host. Using other viral genes, phylogenetic analysis shows these H7N9 isolates splitting off into a separate branch of the family tree. Taken together, these data suggest that these viruses are undergoing genetic changes within the poultry reservoir, and might be on an evolutionary trajectory to adapt to infecting humans with far greater ease.
Using a patient throat swab applied to susceptible cultured cells, a third sequencing experiment on RNA extracted from infected cells was performed. In this case, the data showed that the patient was infected with two influenza A strains, H7N9 and H3N2. Being able to detect simultaneous strains within a sample is critical for understanding the mechanisms of viral reassortment from which new strains emerge.
Early Recognition of Outbreaks
For genetic epidemiology, rapid and accurate next-generation sequencing technology provides high resolution data for the identification, classification, and transmission path investigation of viruses or any other microbial pathogen capable of producing the next pandemic.
The successes of the dedicated Chinese researchers at the Jiangsu CDC illustrate the utility of next-generation sequencing for identifying possible virus transmission routes in the field. More studies from public health agencies will hopefully lead to a better understanding of influenza virus evolution and transmission to help control human infection, and demonstrate the effectiveness of NGS as tool for infectious disease surveillance.
Zhu Y, Qi Z, Cui L, Zhou M, Wang H. (2013) Human co-infection with novel avian influenza A H7N9 and influenza A H3N2 viruses in Jiangsu province, China. The Lancet (381)9883 2134.
Bao C-J, Cui L-B, Zhou M-H, Hong L, Gao GF, et al. (2013) Live-animal markets and influenza A (H7N9) virus infection. N Engl J Med (368) 2337-2339.