An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 ...serotype neuraminidase （NA） has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins （HAs） have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/ Shanghai/I/2013. NAs from these two viruses are distinct as the A/Shanghai/I/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance （over 100 000-fold） using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 （R294-containing） and A/Shanghai/I/2013 N9 （K294-containing）. R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.
To determine the seroprevalence and seroconversion of avian influenza virus (AIV) antibodies in poultry workers, we conducted a seroepidemiologic study in 7 areas of China during December 2014-April ...2016. We used viral isolation and reverse transcription PCR to detect AIVs in specimens from live poultry markets. We analyzed 2,124 serum samples obtained from 1,407 poultry workers by using hemagglutination inhibition and microneutralization assays. We noted seroprevalence of AIV antibodies for subtypes H9N2, H7N9, H6N1, H5N1-SC29, H5N6, H5N1-SH199, and H6N6. In serum from participants with longitudinal samples, we noted seroconversion, with >4-fold rise in titers, for H9N2, H7N9, H6N1, H5N1-SC29, H6N6, H5N6, and H5N1-SH199 subtypes. We found no evidence of H10N8 subtype. The distribution of AIV antibodies provided evidence of asymptomatic infection. We found that AIV antibody prevalence in live poultry markets correlated with increased risk for H7N9 and H9N2 infection among poultry workers.
Sequencing of Ebola virus (EBOV) genomes during the 2014-2016 epidemic identified several naturally occurring, dominant mutations potentially impacting virulence or tropism. In this study, we ...characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA polymerase (L). Compared with the wild-type (WT) EBOV C07 isolate, NP and L mutants conferred a replication advantage in monkey Vero E6, human A549, and insectivorous bat Tb1.Lu cells, while L mutants displayed a disadvantage in human Huh7 cells. The replication of the GP mutant was significantly delayed in Tb1.Lu cells and similar to that of the WT in other cells. The L mutant was less virulent, as evidenced by increased survival for mice and a significantly delayed time to death for ferrets, but increased lengths of the period of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework for the study of other mutations.
During the Ebola virus (EBOV) disease outbreak in West Africa in 2014-2016, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein GP, nucleoprotein NP, or RNA-dependent RNA polymerase L) in cell culture, as well as in mice and ferrets, by generating recombinant viruses (based on an early West African EBOV strain) each carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas the GP mutation slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.
Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of ...avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China.
We recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens.
We isolated Tamdy virus (TAMV; strain XJ01/TAMV/China/2018) from Hyalomma asiaticum ticks infesting Bactrian camels in Xinjiang, China, in 2018. The genome of the strain showed high nucleotide ...similarity with previously described TAMV strains from Asia. Our study highlights the potential threat of TAMV to public health in China.
Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit ...vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced T
H
1/T
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2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 μg or 50 μg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.
Primary amoebic meningoencephalitis (PAM), caused by Naegleria fowleri, is a rare protozoan infectious disease in China. A fatality rate of over 95% had been reported due to extremely rapid disease ...progression in the USA and other countries. Rapid and precise identification of the causative agent is very important to clinicians for guiding their choices for administering countermeasures in the clinic. In this report, we applied the next-generation sequencing (NGS) method to rapidly show that N. fowleri was the causative agent of a fatal case involving a 42-year-old man with severe PAM disease, the first reported in mainland China.
A 42-year old male in a deep coma was admitted to Shenzhen Third People's Hospital, a special medical care unit with expertise in infectious diseases. Increased intracranial pressure was detected. The cerebrospinal fluid (CSF) sample was found to be red and cloudy with increased leukocyte and protein levels. While bacterial cultures with CSF were negative, N. fowleri was determined to be the causative agent with NGS. Amphotericin B (AmB), a drug with anti-amoeba activity, was used immediately, but the treatment came too late and the patient died 2 days after the NGS confirmation.
In this paper, we reported a case of PAM disease for the first time in mainland China. NGS was used for rapid diagnosis and provided guidance for prescribing medications. However, the patient died due to a late admission amid advanced PAM disease. Early detection of N. fowleri is necessary in order to select effective drug treatments and control the disease progression. Despite the negative survival outcome, NGS was shown to be a promising method of rapid and precise identification of N. fowleri.
Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,600 human infections, posing a threat to public health. An emerging concern is whether H7N9 AIVs will cause pandemics among ...humans. Molecular analysis of hemagglutinin (HA), which is a critical determinant of interspecies transmission, shows that the current H7N9 AIVs are still dual-receptor tropic, indicating limited human-to-human transmission potency. Mutagenesis and structural studies reveal that a G186V substitution is sufficient for H7N9 AIVs to acquire human receptor-binding capacity, and a Q226L substitution would favor binding to both avian and human receptors only when paired with A138/V186/P221 hydrophobic residues. These data suggest a different evolutionary route of H7N9 viruses compared to other AIV-subtype HAs.
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•G186V mutation is key for H7N9 HA to acquire human receptor-binding capacity•Q226L mutation favors receptor binding only within a specific microenvironment•G186V mutation might be selected earlier than Q226L in evolution•Structural basis for G186V and Q226L mutation affecting the receptor tropism
Residue substitution in hemagglutinin (HA) could be a determinant of interspecies transmission of influenza virus. Xu et al. report a unique evolutionary route behind the avian-to-human receptor-binding adaptation in H7N9-subtype HA.
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