Xianqiao Zeng

Continuing Reassortment Leads to the Genetic Diversity of Influenza Virus H7N9 in Guangdong, China

ABSTRACT On 30 March 2013, a novel avian influenza A H7N9 virus causing severe human respiratory infections was identified in China. Preliminary sequence analyses have shown that the virus is a reassortant of H7N9 and H9N2 avian influenza viruses. In this study, we conducted enhanced surveillance for H7N9 virus in Guangdong, China, from April to August 2013. We isolated two H7N9 viral strains from environmental samples associated with poultry markets and one from a clinical patient. Sequence analyses showed that the Guangdong H7N9 virus isolated from April to May shared high sequence similarity with other strains from eastern China. The A/Guangdong/1/2013 (H7N9) virus isolated from the Guangdong patient on 10 August 2013 was divergent from previously sequenced H7N9 viruses and more closely related to local circulating H9N2 viruses in the NS and NP genes. Phylogenetic analyses revealed that four internal genes of the A/Guangdong/1/2013 (H7N9) virus—the NS, NP, PB1, and PB2 genes—were in clusters different from those for H7N9 viruses identified previously in other provinces of China. The discovery presented here suggests that continuing reassortment led to the emergence of the A/Guangdong/1/2013 (H7N9) virus as a novel H7N9 virus in Guangdong, China, and that viral adaptation to avian and human hosts must be assessed. IMPORTANCE In this study, we isolated and characterized the avian influenza A H7N9 virus in Guangdong, China, from April to August 2013. We show that the viruses isolated from Guangdong environmental samples and chickens from April to May 2013 were highly similar to other H7N9 strains found in eastern China. The H7N9 virus isolated from the clinical patient in Guangdong in August 2013 was divergent from previously identified H7N9 viruses, with the NS and NP genes originating from recent H9N2 viruses circulating in the province. This study provides direct evidence that continuing reassortment occurred and led to the emergence of a novel H7N9 influenza virus in Guangdong, China. These results also shed light on how the H7N9 virus evolved, which is critically important for future monitoring and tracing of viral transmission.

Circulation of Reassortant Influenza A(H7N9) Viruses in Poultry and Humans, Guangdong Province, China, 2013

Influenza A(H7N9) virus emerged in eastern China in February 2013 and continues to circulate in this region, but its ecology is poorly understood. In April 2013, the Guangdong Provincial Center for Disease Control and Prevention (CDC) implemented environmental and human syndromic surveillance for the virus. Environmental samples from poultry markets in 21 city CDCs (n = 8,942) and respiratory samples from persons with influenza-like illness or pneumonia (n = 32,342) were tested; viruses isolated from 6 environmental samples and 16 patients were sequenced. Sequence analysis showed co-circulation of 4 influenza A(H7N9) virus strains that evolved by reassortment with avian influenza A(H9N2) viruses circulating in this region. In addition, an increase in human cases starting in late 2013 coincided with an increase in influenza A H7 virus isolates detected by environmental surveillance. Co-circulation of multiple avian influenza viruses that can infect humans highlights the need for increased surveillance of poultry and potential environmental sources.

Emergence and Continuous Evolution of Genotype 1E Rubella Viruses in China

ABSTRACT In China, rubella vaccination was introduced into the national immunization program in 2008, and a rubella epidemic occurred in the same year. In order to know whether changes in the genotypic distribution of rubella viruses have occurred in the postvaccination era, we investigate in detail the epidemiological profile of rubella in China and estimate the evolutionary rate, molecular clock phylogeny, and demographic history of the predominant rubella virus genotypes circulating in China using Bayesian Markov chain Monte Carlo phylodynamic analyses. 1E was found to be the predominant rubella virus genotype since its initial isolation in China in 2001, and no genotypic shift has occurred since then. The results suggest that the global 1E genotype may have diverged in 1995 and that it has evolved at a mutation rate of 1.65 × 10−3 per site per year. The Chinese 1E rubella virus isolates were grouped into either cluster 1 or cluster 2, which likely originated in 1997 and 2006, respectively. Cluster 1 viruses were found in all provinces examined in this study and had a mutation rate of 1.90 × 10−3 per site per year. The effective number of infections remained constant until 2007, and along with the introduction of rubella vaccine into the national immunization program, although the circulation of cluster 1 viruses has not been interrupted, some viral lineages have disappeared, and the epidemic started a decline that led to a decrease in the effective population size. Cluster 2 viruses were found only in Hainan Province, likely because of importation.

Family clusters of avian influenza A H7N9 virus infection in Guangdong Province, China.

ABSTRACT Since its first identification, the epizootic avian influenza A H7N9 virus has continued to cause infections in China. Two waves were observed during this outbreak. No cases were reported from Guangdong Province during the first wave, but this province became one of the prime outbreak sites during the second wave. In order to identify the transmission potential of this continuously evolving infectious virus, our research group monitored all clusters of H7N9 infections during the second wave of the epidemic in Guangdong Province. Epidemiological, clinical, and virological data on these patients were collected and analyzed. Three family clusters including six cases of H7N9 infection were recorded. The virus caused severe disease in two adult patients but only mild symptoms for all four pediatric patients. All patients reported direct poultry or poultry market exposure history. Relevant environment samples collected according to their reported exposures tested H7N9 positive. Virus isolates from patients in the same cluster shared high sequence similarities. In conclusion, although continually evolving, the currently circulating H7N9 viruses in Guangdong Province have not yet demonstrated the capacity for efficient and sustained person-to-person transmission.

Isolation of H5N6, H7N9 and H9N2 avian influenza A viruses from air sampled at live poultry markets in China, 2014 and 2015

Zoonotic infections by avian influenza viruses occur at the human–poultry interface, but the modes of transmission have not been fully investigated. We assessed the potential for airborne and fomite transmission at live poultry markets in Guangzhou city and in Hong Kong Special Administrative Region (SAR), China, during 2014 and 2015. Viral genome and infectious avian influenza A viruses of H5N6, H7N9, and H9N2 subtypes were detected predominantly from particles larger or equal to 1 μm in diameter in the air sampled with cyclone-based bioaerosol samplers at the live poultry markets in Guangzhou. Influenza A(H9N2) viruses were ubiquitously isolated every month during the study period from air and environmental swabs, and different lineages of H9N2 virus were isolated from markets where chickens and minor land-based poultry were sold. The use of de-feathering devices increased the quantity of virus-laden airborne particles while market closure reduced the amount of such particles. The results highlight the possibility of airborne transmission of avian influenza viruses among poultry or from poultry to humans within such settings. This may explain epidemiological observations in which some patients with H7N9 infection reported being in markets but no direct contact with live poultry or poultry stalls.

Reassortment of Avian Influenza A/H6N6 Viruses from Live Poultry Markets in Guangdong, China.

Since early 2013, H7N9-subtype avian influenza virus (AIV) has caused human infection in eastern China. To evaluate AIV contamination and the public risk of infection, we systematically implemented environmental sampling from live poultry markets in Guangdong Province. Through real-time polymerase chain reaction assays and next-generation sequencing, we generated full nucleotide sequences of all 10 H6N6 AIVs isolated during sampling. Focusing on sequence analyses of hemagglutinin genes of the 10 H6N6 AIVs revealed that the viruses were low pathogenic AIVs with the typical hemagglutinin cleavage site of P-Q-I-E-T-R-G. The hemagglutinin, neuraminidase, and nucleocapsid genes of nine AIVs were of ST2853-like (H6-subtype) lineage, ST192-like (N6-subtype) lineage, and HN573-like (H6-subtype) lineage, respectively; whereas the other five genes were of ST339-like (H6-subtype) lineage. However, the polymerase PB2 and nucleocapsid genes of one strain (HZ057) were of GS/GD-like (H5N1-subtype) and ST339-like lineages. Phylogenic analysis revealed that all eight genes of the 10 viruses belonged to Eurasian avian lineage. Altogether, the 10 AIVs were reassortants of different genetic groups of exchanges with the same virus subtype, thus illustrating the genetic diversity and complexity of H6N6-subtype AIVs in Guangdong Province.

Effect of live poultry market interventions on influenza A(H7N9) virus, Guangdong, China

Temporary closure of these markets appears not to have halted virus transmission or prevented its dissemination.

Mild influenza A/H7N9 infection among children in Guangdong Province.

We describe the clinical and epidemiologic characteristics of 7 children infected with A/H7N9 in Guangdong Province during the winter of 2013–2014. Our results indicate that the mild or asymptomatic characteristics common in H7N9-infected children could pose challenges to our surveillance system becoming a hidden threat to the public health of China and the world.

Serologic screenings for H7N9 from three sources among high-risk groups in the early stage of H7N9 circulation in Guangdong Province, China

BackgroundThe aim of this study was to assess the prevalence of the novel avian influenza A virus (H7N9) in three high risk groups. The groups were divided into those exposed through infected individuals, those exposed through poultry and those individuals exposed through the external environment, in the early stage of the epidemic in Guangdong Province, which is located in the southern region of China.MethodsSerologic studies were conducted among samples collected from individuals who had close contact with the first H7N9 infected patient reported in Guangdong Province, those who were most likely exposed to the first group of H7N9 infected poultry, and those who might have been exposed to H7N9 in the environmental settings, namely hemagglutinin inhibition (HI) and microneutralizaiton(MN) assays using three viruses as antigens.ResultsThe alignment results of the viral sequences indicated the similarity of the HA gene sequence among viruses from exposure to infected poultry, infected humans and contaminated environments were highly conserved. Seven samples of individuals exposed to contaminated environments were positive in the HI assay and one sample among them was positive in the MN assay using poultry H7N9 virus as the antigen. One sample was positive against human H7N9 virus and 3 samples were positive against environmental H7N9 among those that were in contact with infected patients in HI assay. None of these were positive in MN assay. All HI titers of the 240 samples from those individuals in contact with infected poultry were less than 40 aganist the antigens from three viruses.ConclusionsThe results suggest that when the H7N9 virus was in the early stages of circulation in Guangdong Province, the antigenic sites of the HA proteins of the H7N9 strain isolated from different hosts were highly conserved. The risk of new infection is low in individuals who have contact with the infected patients, poultry or a contaminated environment in the early stages of the circulation of the H7N9 virus.

Genetic Changes of Reemerged Influenza A(H7N9) Viruses, China

To the Editor: From March 30, 2013, through April 8, 2014, a total of 401 human infections with novel avian influenza A (H7N9) virus were reported in China (1). In the initial wave from February through May 2013, cases were laboratory confirmed for 133 patients (45 died), mainly in eastern China. From June through early October 2013, only 2 laboratory-confirmed cases were reported in China. One of these, identified on August 10, 2013, was the first case of influenza A(H7N9) virus infection in Guangdong Province (strain A/Guangdong/HZ-01/2013). However, a second wave of influenza A(H7N9) virus infection began on October 14, 2013 (2). As of April 8, 2014, a total of 266 laboratory-confirmed cases had been reported, mainly in Zhejiang Province in eastern China (92 cases, 37 deaths) and Guangdong Province in southern China (99 cases, 30 deaths). Previous sequencing studies suggested that 6 of the 8 influenza A(H7N9) virus RNA segments were acquired from influenza A(H9N2) virus. This acquisition process involved at least 2 steps of sequential reassortment; the most recent event most likely occurred in the Yangtze River Delta area of eastern China (3–5). To date, nearly all analyses have been performed by using sequences obtained from viruses isolated during the first wave of infection; changes associated with viruses isolated during the second wave are largely unknown (6). We therefore conducted phylogenetic analyses of whole-genome sequence data for 15 influenza A(H7N9) viruses isolated from human patients in Guangdong from November 4, 2013, through January 15, 2014. We estimated maximum-likelihood trees for all 8 RNA segments by using MEGA version 5.2 and the general time-reversible model (7). RNA segments encoding the hemagglutinin, neuraminidase, and matrix genes of A/Guangdong/H7N9 viruses isolated after November 2013 were genetically similar to those of A/Guangdong/HZ-01/2013 and H7N9 strains from the first wave of influenza (Technical Appendix). An additional 4 RNA segments (nonstructural protein [NS], nucleocapsid protein [NP], polymerase basic proteins [PB] 1 and 2) of A/Guangdong/H7N9 influenza viruses isolated after November 2013 were clustered with A/Guangdong/HZ-01/2013 virus and were divergent from all currently sequenced subtype H7N9 viruses from the first wave in eastern China. The only exception was the NP segment of A/Guangdong/SZ-026/2014, which was found segregated into a separate cluster with subtype H9N2 viruses from Shandong Province. Moreover, analyses showed that RNA segments encoding NS, NP, PB1, and PB2 of A/Guangdong/H7N9 isolated after November 2013 were most similar to the same segments from influenza A(H9N2) viruses that had recently circulated in Guangdong (Technical Appendix Figure, panels D–G). That is, NS, NP, PB1, and PB2 showed greater similarity to local subtype H9N2 viruses from Guangdong than to subtype H7N9 viruses from the first wave of influenza. Notably, 2 separate clusters were observed for the phylogenetic tree of the RNA segment encoding the polymerase acidic gene (Technical Appendix Figure, panel H). A/Guangdong/HZ-01/2013-like viruses clustered with subtype H7N9 viruses from the first wave of influenza. However, A/Guangdong/DG-02/2013-like viruses were clustered with subtype H9N2 influenza viruses circulating in Guangdong, suggesting that recent reassortment with circulating subtype H9N2 viruses occurred after the first case of infection with influenza A(H7N9) virus reported in Guangdong (Technical Appendix Figure, panel H). This study provides evidence that influenza A(H7N9) viruses isolated during the second wave of influenza in Guangdong differ genetically (in 5 of the 8 RNA segments) from that of influenza A(H7N9) viruses isolated during the first wave. High similarity of these 5 segments with those of locally circulating subtype H9N2 viruses suggests that rapid and continued reassortment with circulating subtype H9N2 viruses occurred during the second wave of the influenza A(H7N9) virus epidemic. Because reassortment and genetic changes can contribute to host fitness and infection capacity of reemerged influenza A(H7N9) viruses, studies of pathogenicity and transmission, to reveal the exact role of each genetic alteration, are needed. Technical Appendix: Phylogenetic tree of the 8 RNA segments from influenza A(H7N9) virus isolates. Click here to view.(174K, pdf)