Hanzhong Ni

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.

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.

Continuing Reassortant of H5N6 Subtype Highly Pathogenic Avian Influenza Virus in Guangdong.

First identified in May 2014 in Chinas Sichuan Province, initial cases of H5N6 avian influenza virus (AIV) infection in humans raised great concerns about the viruss prevalence, origin, and development. To evaluate both AIV contamination in live poultry markets (LPMs) and the risk of AIV infection in humans, we have conducted surveillance of LPMs in Guangdong Province since 2013 as part of environmental sampling programs. With environmental samples associated with these LPMs, we performed genetic and phylogenetic analyses of 10 H5N6 AIVs isolated from different cities of Guangdong Province from different years. Results revealed that the H5N6 viruses were reassortants with hemagglutinin (HA) genes derived from clade 2.3.4.4 of H5-subtype AIV, yet neuraminidase (NA) genes derived from H6N6 AIV. Unlike the other seven H5N6 viruses isolated in first 7 months of 2014, all of which shared remarkable sequence similarity with the H5N1 AIV in all internal genes, the PB2 genes of GZ693, GZ670, and ZS558 more closely related to H6N6 AIV and the PB1 gene of GZ693 to the H3-subtype AIV. Phylogenetic analyses revealed that the environmental H5N6 AIV related closely to human H5N6 AIVs isolated in Guangdong. These results thus suggest that continued reassortment has enabled the emergence of a novel H5N6 virus in Guangdong, as well as highlight the potential risk of highly pathogenic H5N6 AIVs in the province.

Genetic mutations in influenza H3N2 viruses from a 2012 epidemic in Southern China

BackgroundAn influenza H3N2 epidemic occurred throughout Southern China in 2012.MethodsWe analyzed the hemagglutinin (HA) and neuraminidase (NA) genes of influenza H3N2 strains isolated between 2011–2012 from Guangdong. Mutation sites, evolutionary selection, antigenic sites, and N-glycosylation within these strains were analyzed.ResultsThe 2011–2012 Guangdong strains contained the HA-A214S, HA-V239I, HA-N328S, NA-L81P, and NA-D93G mutations, similar to those seen in the A/ Perth/16/2009 influenza strain. The HA-NSS061–063 and NNS160–162 glycosylation sites were prevalent among the 2011–2012 Guangdong strains but the NA-NRS402–404 site was deleted. Antigenically, there was a four-fold difference between A/Perth/16/2009 -like strains and the 2011–2012 Guangdong strains.ConclusionAntigenic drift of the H3N2 subtype contributed to the occurrence of the Southern China influenza epidemic of 2012.

Serologic survey of the pandemic H1N1 2009 virus in Guangdong Province, China: a cross sectional study.

Background Relying on surveillance of clinical cases limits the ability to understand the full impact and severity of an epidemic, which urges a deep insight into the serological evidence of infection and transmission feature of pandemic H1N1 2009 (pH1N1) virus in Guangdong province. Methods In this cross-sectional serological survey, serum samples were collected by multi-stage stratified random sampling in Jan 2010. Antibody titers were measured by hemagglutination inhibition (HI) assay. Age-specific and region-specific prevalence were calculated based on the results of HI assay (positive, HI titer≥1∶40). Results A total of 4,319 serum samples had been collected from subjects without vaccination with pH1N1 vaccine. The seroprevalence was 22.82% (985/4,319). By contrast, there was a marked spatial heterogeneity in prevalence. The seroprevalence was 27.3% in large city, 21.4% in medium cities, higher than that of 20.2% in rural areas. The seroprevalence was highest in 11–20 age group (32.8%), however, in those above 60 years of age group, which was 12.6%, lower than other age groups. On the other hand, antibody titers to pH1N1 virus were highest in school children, which were followed by a gradual decrease in adult. However, in the elderly groups from cities, especially from large city, the antibody titer to pH1N1 increased significantly and reached a much higher level. Conclusion Our results showed that the prevalence for pH1N1 was correlated with age and population density. Preexisting antibody may have protected the very old from pH1N1 infection, while original antigenic sin and immunosenescence may have contributed to greater severity once infected. These should be considered when studying the pathogenesis and transmission of influenza virus and formulating strategies on vaccination and treatment.

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.

Adamantane- and Oseltamivir-Resistant Seasonal A(H1N1) and Pandemic A(H1N1) 2009 Influenza Viruses in Guangdong, China, During 2008 and 2009

ABSTRACT Adamantane and oseltamivir resistance among influenza viruses is a major concern to public health officials. To determine the prevalence of antiviral-resistant influenza viruses in Guangdong, China, 244 seasonal A (H1N1) and 222 pandemic A (H1N1) 2009 viruses were screened for oseltamivir resistance by a fluorescence-based neuraminidase (NA) inhibition assay along with NA gene sequencing. Also, 147 seasonal A (H1N1) viruses were sequenced to detect adamantane resistance markers in M2. Adamantane-resistant seasonal A (H1N1) viruses clustering to clade 2C were dominant in 2008, followed by oseltamivir-resistant seasonal A (H1N1) viruses, clustering to clade 2B during January and May 2009. In June 2009, a lineage of double-resistant seasonal A (H1N1) viruses emerged, until it was replaced by the pandemic A (H1N1) 2009 viruses. The lineage most likely resulted from reassortment under the pressure of the overuse of adamantanes. As all viruses were resistant to at least one of the two types of antiviral agents, the need for close monitoring of the prevalence of antiviral resistance is stressed.

Temporal trends of influenza A (H1N1) virus seroprevalence following 2009 pandemic wave in Guangdong, China: three cross-sectional serology surveys.

Background To evaluate the temporal trends of seroprevalence to pH1N1 among the Guangdong population following 2009 H1N1 pandemic wave, we conducted three cross-sectional serology surveys in 2010. Methodology/Principal Findings Three surveys were carried out consecutively in 2010 from January 8 to January 24, from March 15 to April 10 and from August 23 to September 4. Sample populations comprising of 4725, 4727, and 4721 subjects respectively were randomly selected for study in these three surveys. The level of antibodies against pH1N1 was evaluated by hemagglutination inhibition assay. In survey 1, the seroprevalence of pH1N1 among all the subjects is 25.1%, declining to 18.4% in survey 2 and increasing to 21.4% in survey 3. Among vaccinated subjects, the seroprevalence was 49.0%, 53.0%, and 49.4% in the three consecutive surveys, showing no significant differences. In contrast, among non-vaccinated subjects, the seroprevalence declined significantly from 22.8% (survey 1) to 14.3% (survey 2) and subsequently increased to 18.1% (survey 3). The multivariate logistic regression analysis revealed that seroprevalence to pH1N1 in non-vaccinated individuals correlated with the investigated order of the surveys, age, and region (all P<0.05). However, it was not correlated with gender (P = 0.650), seasonal influenza vaccination history (P = 0.402) and symptoms (P = 0.074). Conclusions/Significance In Guangdong, the seroprevalance to pH1N1 decreased initially and then rebounded modestly during the first 9 months following the 2009 pandemic wave. Our results suggest that the prevalence of pH1N1 is still correlated with age and population density during the post-pandemic period. An early end to the free pH1N1 vaccination program might be another important reason for the slight rebound in seroprevalance. Our study findings can help the Guangdong authorities to make evidence-based decisions about a long-term vaccination strategy and boost immunity in specific population groups (such as children and people living in the capital-city) to prevent further transmission in the future.