Xiaohui Zou

Human infection with a novel, highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings.

BACKGROUND AND OBJECTIVES Severe infection with avian influenza A (H5N6) virus in humans was identified first in 2014 in China. Before that, it was unknown or unclear if the disease or the pathogen affected people. This study illustrates the virological and clinical findings of a fatal H5N6 virus infection in a human patient. METHODS We obtained and analyzed the clinical, epidemiological, and virological data from the patient. Reverse transcription polymerase chain reaction (RT-PCR), viral culture, and sequencing were conducted for determination of the causative pathogen. RESULTS The patient, who presented with fever, severe pneumonia, leucopenia, and lymphopenia, developed septic shock and acute respiratory distress syndrome (ARDS), and died on day 10 after illness onset. A novel reassortant avian-origin influenza A (H5N6) virus was isolated from the throat swab or trachea aspirate of the patient. The virus was reassorted with the HA gene of clade 2.3.4.4 H5, the internal genes of clade 2.3.2.1 H5, and the NA gene of the H6N6 avian virus. The cleavage site of the HA gene contained multiple basic amino acids, indicating that the novel H5N6 virus was highly pathogenic in chicken. CONCLUSIONS A novel, highly pathogenic avian influenza H5N6 virus with a backbone of H5N1 virus acquired from the NA gene from the H6N6 virus has been identified. It caused human infection resulting in severe respiratory disease.

Genetic Diversity of Avian Influenza A (H10N8) Virus in Live Poultry Markets and Its Association with Human Infections in China

Following the first human infection with the influenza A (H10N8) virus in Nanchang, China in December 2013, we identified two additional patients on January 19 and February 9, 2014. The epidemiologic, clinical, and virological data from the patients and the environmental specimen collected from 23 local live poultry markets (LPMs) were analyzed. The three H10N8 cases had a history of poultry exposure and presented with high fever (>38°C), rapidly progressive pneumonia and lymphopenia. Substantial high levels of cytokines and chemokines were observed. The sequences from an isolate (A/Environment/Jiangxi/03489/2013 [H10N8]) in an epidemiologically linked LPM showed highly identity with human H10N8 virus, evidencing LPM as the source of human infection. The HA and NA of human and environmental H10N8 isolates showed high identity (99.1–99.9%) while six genotypes with internal genes derived from H9N2, H7N3 and H7N9 subtype viruses were detected in environmental H10N8 isolates. The genotype of the virus causing human infection, Jiangxi/346, possessed a whole internal gene set of the A/Environment/Jiangxi/10618/2014(H9N2)-like virus. Thus, our findings support the notion that LPMs can act as both a gene pool for the generation of novel reassortants and a source for human infection, and intensive surveillance and management should therefore be conducted.

Poultry farms as a source of avian influenza A (H7N9) virus reassortment and human infection

Live poultry markets are a source of human infection with avian influenza A (H7N9) virus. On February 21, 2014, a poultry farmer infected with H7N9 virus was identified in Jilin, China, and H7N9 and H9N2 viruses were isolated from the patients farm. Reassortment between these subtype viruses generated five genotypes, one of which caused the human infection. The date of H7N9 virus introduction to the farm is estimated to be between August 21, 2013 (95% confidence interval [CI] June 6, 2013-October 6, 2013) and September 25, 2013 (95% CI May 28, 2013-January 4, 2014), suggesting that the most likely source of virus introduction was the first batch of poultry purchased in August 2013. The reassortment event that led to the human virus may have occurred between January 2, 2014 (95% CI November 8, 2013-February 12, 2014) and February 12, 2014 (95% CI January 19, 2014-February 18, 2014). Our findings demonstrate that poultry farms could be a source of reassortment between H7N9 virus and H9N2 virus as well as human infection, which emphasizes the importance to public health of active avian influenza surveillance at poultry farms.

Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses

ABSTRACT Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans. IMPORTANCE Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.

Reassortant Eurasian Avian-Like Influenza A(H1N1) Virus from a Severely Ill Child, Hunan Province, China, 2015

Infectivity and virulence of this virus in mice are higher than for previous human-origin Eurasian avian–like viruses.

Post-mortem findings in a patient with avian influenza A (H5N6) virus infection

Avian influenza A (H5N6) has been found to infect humans, and has resulted in ten cases with six deaths in China since 2014. Here, we describe the systematic post-mortem pathology of a patient fatally infected with H5N6 virus and evaluate the associated pathogenesis compared with H1N1 pdm09 fatal cases. The most prominent histopathological features were diffuse alveolar damage and pulmonary vasculitis in the lungs of the patient. The virus disseminated to extrapulmonary organs, including the brain. Compared with H1N1 pdm09 fatal infection, H5N6 infection induced a more exacerbated immune response involving overt pulmonary inflammation, which led to alveolar damage and respiratory failure.

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses’ role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

OBJECTIVE To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform. METHODS Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. RESULTS The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus. CONCLUSION The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

The repeated introduction of the H3N2 virus from human to swine during 1979-1993 in China.

Limited data are available regarding the swine influenza viruses (SIVs) that circulated in Mainland China prior to the 1990s. Eleven H3N2 virus strains were isolated from swine populations from 1979 to 1992. To determine the origin and tendency of these SIVs, the phylogenetic and antigenic properties of these viruses were analyzed based on the whole genome sequenced and the HI titrations with post-infection ferret antisera against influenza A (H3N2) virus isolates of swine and human origin. The results revealed that these 11 SIVs originated from humans and were not maintained in swine populations, indicating the interspecies transmission from humans to pigs occurred frequently and independently throughout these periods. However, human H3N2 viruses might not have the ability to circulate in pig herds.

Fatal Aeromonas bacteraemia in West Africa.

We read with interest the communication by Batra and collegues who described nosocomial infections due to Aeromonas species in trauma patients. Typically, Aeromonas infection results in a mild illness in immunocompetent humans. In contrast, severe clinical complications occur in immunocompromised patients, such as hepatobiliary infections, primary bacteremia, meningitis, and endocarditis. Fatal cases of Aeromonas infections have been reported sporadically, especially in patients had septicemia developed, with fatal outcomes occurring 2e4 days postAeromonas infection. Here, we identified a large amount of Aeromonas sequences in the blood sample of a dead case from western Africa by unbiased metagenomic sequencing, indicating the Aeromonas might be associated with the death of the patient. The patient was a 55-year-old man from Mali, which is located in western Africa. He arrived in China on 11 October 2014 for training purposes. The second evening after his arrival, a fever developed, followed by sputum production, frequent urination, dysuria, and diarrhea, but without abdomen pain. Fevers and chills developed the subsequent morning, while mental confusion was noted at 11:00 am on the 3rd day. On 13 October, the patient was admitted to the hospital with a body temperature of 38.5 C, which increased to 39.3 C after 8 h. The physical examination revealed a normal blood pressure, but tachycardia at 137 beats per minute. Mental confusion with barylalia was noted. Coarse breath sounds were auscultated on examination of the lungs, but no rales. Laboratory testing revealed white and red blood cell counts of 27.58 10/L (normal range, 4.0e10 10/L) and 2.79 10/L (normal range, 4.2e5.9 10/L), respectively. The hemoglobin was 82 g/L (normal range, 120e160 g/L), the percentage neutrophils was 82.3%, the platelet count was 86 10/L (normal range, 150e350 10/L), and the CRP level was 177.80 mg/L. Increased texture and opacity were shown in the lung fields bilaterally on chest radiography. Other observations included a possible lung infection, a left pleural