Outdoor PM2.5 concentration and rate of change in COVID-19 infection in provincial capital cities in China.

This study investigates thoroughly whether acute exposure to outdoor PM2.5 concentration, P, modifies the rate of change in the daily number of COVID-19 infections (R) across 18 high infection provincial capitals in China, including Wuhan. A best-fit multiple linear regression model was constructed to model the relationship between P and R, from 1 January to 20 March 2020, after accounting for meteorology, net move-in mobility (NM), time trend (T), co-morbidity (CM), and the time-lag effects. Regression analysis shows that P (ß = 0.4309, p < 0.001) is the most significant determinant of R. In addition, T (ß = -0.3870, p < 0.001), absolute humidity (AH) (ß = 0.2476, p = 0.002), P × AH (ß = -0.2237, p < 0.001), and NM (ß = 0.1383, p = 0.003) are more significant determinants of R, as compared to GDP per capita (ß = 0.1115, p = 0.015) and CM (Asthma) (ß = 0.1273, p = 0.005). A matching technique was adopted to demonstrate a possible causal relationship between P and R across 18 provincial capital cities. A 10 µg/m3 increase in P gives a 1.5% increase in R (p < 0.001). Interaction analysis also reveals that P × AH and R are negatively correlated (ß = -0.2237, p < 0.001). Given that P exacerbates R, we recommend the installation of air purifiers and improved air ventilation to reduce the effect of P on R. Given the increasing observation that COVID-19 is airborne, measures that reduce P, plus mandatory masking that reduces the risks of COVID-19 associated with viral-particulate transmission, are strongly recommended. Our study is distinguished by the focus on the rate of change instead of the individual cases of COVID-19 when modelling the statistical relationship between R and P in China; causal instead of correlation analysis via the matching analysis, while taking into account the key confounders, and the individual plus the interaction effects of P and AH on R.

Adaptation of avian influenza virus to a swine host.

The emergence of pathogenic RNA viruses into new hosts can have dramatic consequences for both livestock and public health. Here we characterize the viral genetic changes that were observed in a previous study which experimentally adapted a field isolate of duck influenza virus to swine respiratory cells. Both pre-existing and de novo mutations were selected during this adaptation. We compare the in vitro growth dynamics of the adapted virus with those of the original strain as well as all possible reassortants using reverse genetics. This full factorial design showed that viral gene segments are involved in complex epistatic interactions on virus fitness, including negative and sign epistasis. We also identify two point mutations at positions 67 and 113 of the HA2 subunit of the hemagglutinin protein conferring a fast growth phenotype on the naïve avian virus in swine cells. These HA2 mutations enhance the pH dependent, HA-mediated membrane fusion. A global H1 maximum-likelihood phylogenetic analysis, combined with comprehensive ancestry reconstruction and tests for directional selection, confirmed the field relevance of the mutation at position 113 of HA2. Most notably, this mutation was associated with the establishment of the H1 'avian-like' swine influenza lineage, regarded as the most likely to cause the next influenza pandemic in humans. This multidisciplinary approach to study the genetics of viral adaptation provides unique insights on the underlying processes leading to influenza emergence in a new host species, and identifies specific targets for future surveillance and functional studies.

[Expression of neuraminidase gene of influenza virus H1N1 in baculovirus-expression system].

OBJECTIVE: To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus. METHODS: Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA. RESULTS: PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8), indicating high antigenicity. CONCLUSION: Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.

Evaluation of the immune response to recombinant DNA vaccine and adenoviral vaccine co-expressing the M1 and HA genes of H5N1 influenza virus in mice.

In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.

[Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli].

OBJECTIVE: To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein. METHODS: M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1. RESULTS: The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals. CONCLUSION: These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.

[Construction of vectors expressing M2 and NA genes of H5N1 influenza virus].

OBJECTIVE: To construct vectors expressing M2 and NA genes of H5N1 influenza virus. METHODS: Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids. RESULTS: Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA. CONCLUSION: Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.

[Establishment of a stable and inducible mammalian cell line expressing influenza virus A M2 protein].

Matrix protein 2(M2) is an integral tetrameric membrane protein of influenza A virus, which functions as ion channel. M2 sequence has shown remarkable conservation, so there has been growing interest in it as "universal" vaccine. In order to establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator, M2 gene was obtained by PCR amplification from the plasmid containing the segment 7 of influenza A virus strain A/PR/8/34 firstly. The PCR product was cloned into BamH I/Not I restriction site of pcDNA5/FRT/TO vector, and cotransfected with pOG44 which express Flp recombinase into Flp-In T-REx-293 cell. Integration of pcDNA5/FRT/TO-M2 into the cell genome at the Flp Recombination Target (FRT) site brought the SV40 promoter and the initiation codon in frame with the hygromycin resistance gene. Thus, stable cell lines were selected for hygromycin resistance. The expression of M2 protein from hygromycin-resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay (IFA). 16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator. The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.