Mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans.

Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA against contemporary clinical isolates of influenza A and B viruses. The 50 % cellular cytotoxicity (CC50) of MPA in Madin Darby canine kidney cell line was over 50 µM. MPA prevented influenza virus-induced cell death in the cell-protection assay, with significantly lower IC50 for influenza B virus B/411 than that of influenza A(H1N1)pdm09 virus H1/415 (0.208 vs 1.510 µM, P=0.0001). For H1/415, MPA interfered with the early stage of viral replication before protein synthesis. For B/411, MPA may also act at a later stage since MPA was active against B/411 even when added 12 h post-infection. Virus-yield reduction assay showed that the replication of B/411 was completely inhibited by MPA at concentrations ≥0.78 µM, while there was a dose-dependent reduction of viral titer for H1/415. The antiviral effect of MPA was completely reverted by guanosine supplementation. Plaque reduction assay showed that MPA had antiviral activity against eight different clinical isolates of A(H1N1), A(H3N2), A(H7N9) and influenza B viruses (IC50 <1 µM). In summary, MPA has broad-spectrum antiviral activity against human and avian-origin influenza viruses, in addition to its immunomodulatory activity. Together with a high chemotherapeutic index, the use of MPA as an antiviral agent should be further investigated in vivo.

Recombinant influenza A virus hemagglutinin HA2 subunit protects mice against influenza A(H7N9) virus infection.

A novel avian influenza A(H7N9) virus has emerged to infect humans in eastern China since 2013. An effective vaccine is needed because of the high mortality despite antiviral treatment and intensive care. We sought to develop an effective vaccine for A(H7N9) virus. The HA2 subunit was chosen as the vaccine antigen because it is highly conserved among the human A(H7N9) virus strains. Moreover, in silico analysis predicted two immunogenic regions within the HA2 subunit that may contain potential human B-cell epitopes. The HA2 fragment was readily expressed in Escherichia coli. In BALB/c mice, intraperitoneal immunization with two doses of HA2 with imiquimod (2-dose-imiquimod) elicited the highest geometric mean titer (GMT) of anti-HA2 IgG (12699), which was greater than that of two doses of HA2 without imiquimod (2-dose-no-adjuvant) (6350), one dose of HA2 with imiquimod (1-dose-imiquimod) (2000) and one dose of HA2 without imiquimod (1-dose-no-adjuvant) (794). The titer of anti-HA2 IgG was significantly higher in the 1-dose-imiquimod group than the 1-dose-no-adjuvant group. Although both hemagglutination inhibition titers and microneutralization titers were below 10, serum from immunized mice showed neutralizing activity in a fluorescent focus microneutralization assay. In a viral challenge experiment, the 2-dose-imiquimod group had the best survival rate (100 %), followed by the 2-dose-no-adjuvant group (90 %), the 1-dose-imiquimod group (70 %) and the 1-dose-no-adjuvant group (40 %). The 2-dose-imiquimod group also had significantly lower mean pulmonary viral loads than the 1-dose-imiquimod, 1-dose-no-adjuvant and non-immunized groups. This recombinant A(H7N9)-HA2 vaccine should be investigated as a complement to egg- or cell-based live attenuated or subunit influenza vaccines.

Ongoing transmission of avian influenza A viruses in Hong Kong despite very comprehensive poultry control measures: A prospective seroepidemiology study.

OBJECTIVES: Stringent measures have been implemented in Hong Kong to prevent human infections due to avian influenza viruses (AIVs). Here, we report the seroprevalence of AIVs among high risk population. METHODS: In this prospective study, blood samples were collected in October and November 2013 and in July 2014 from workers at live poultry market (LPM) and pig/cattle slaughterhouse (SH) in Hong Kong. Serum antibody titers against A(H5N1), A(H7N9) and A(H9N2) were determined. RESULTS: When an hemagglutination inhibition (HI) titer of 40 was used as the cutoff, the A(H5N1) seropositive rate among LPM workers increased from 0% in 2013 to 37.8% in 2014 (P < 0.001) and the A(H9N2) seropositive rate increased from 10% to 55.6% (P < 0.001). There was no significant increase in A(H7N9) seropositive rate for LPM workers irrespective of cutoff titer. For SH workers, there was no significant increase in HI titer for any AIVs. Significantly more LPM workers had a ≥4-fold increase in A(H5N1) HI titer from 2013 to 2014 than SH workers (60% vs 8.3%, P = 0.020). CONCLUSIONS: There was a significant increase of serum A(H5N1) and A(H9N2) HI titers among Hong Kong LPM workers between 2013 and 2014. Although we cannot exclude some degree of antibody cross-reactivity with other influenza viruses, our results suggest the occurrence of subclinical AIV infections in this population.

Surfactant protein B gene polymorphism is associated with severe influenza.

BACKGROUND: Surfactant proteins play a key role in alveolar stability. We examined whether single nucleotide polymorphisms (SNPs) related to the surfactant protein genes are associated with severe influenza. METHODS: In the first cohort, 12 SNPs related to surfactant protein genes were compared between Chinese patients with severe and mild pandemic 2009 influenza A(H1N1) (A[H1N1]pdm09) infection who were matched for age, sex, and underlying risk conditions. The SNP rs1130866, which was significantly different between the two groups, was further genotyped in a second cohort of patients. Multivariate analysis was performed to control for confounding factors. The genotype frequencies were also compared with those of the general Han Chinese population. RESULTS: This study consisted of 380 patients with A(H1N1)pdm09 infection. In the first cohort of 84 patients, the C allele of rs1130866, an SNP in the surfactant protein B gene (SFTPB), was significantly associated with severe disease (OR = 3.37, P = .0048), although the P value was .057 after Bonferroni correction. In the second cohort of 296 patients, the C/C genotype was confirmed in the univariate analysis to be associated with severe disease. Multivariate analysis of the second cohort showed that genotype C/C was an independent risk factor for severe A(H1N1)pdm09 infection (second cohort: OR = 2.087, P = .023). Compared to the general Han Chinese population, the C/C genotype was overrepresented in patients with severe A(H1N1)pdm09 infection (OR = 3.232, P = .00000056). CONCLUSIONS: SFTPB polymorphism is associated with severe influenza. The role of SFTPB in influenza warrants further studies.

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be emphasized in the investigation of disease outbreaks in human and animals.