Molecular epidemiology and evolution of A[H1N1] pdm09 and H3N2 viruses in Jordan, 2011-2013

Understanding the genetic evolution of A [H1N1]pdm09 and H3N2 viruses can help better select strains to be included in the annual influenza vaccine. There is little information on their evolution in Jordan so this study investigated the genetic and antigenic variability of A[H1N1]pdm09 and H3N2 viruses in Jordan by performing phylogenetic and genetic analyses of the HA and NA genes of A[H1N1]pdm09 and H3N2 viruses between 2011 and 2013. The full HA and NA genes of 16 H1N1-positive samples obtained in our study and 21 published HA sequences and 20 published NA sequences from Jordanian viruses that were available on online gene databases were analysed. For H3N2, we generated 20 HA and 19 NA sequences and included 19 published HA and NA sequences each in the analysis. Jordanian H1N1 viruses had mutations that are characteristic of antigenic group 6 while H3N2 virus mutations belonged to group 3. No markers of resistance to oseltamivir were detected. The individual mutations are described in detail

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.

Occurrence of swine influenza virus infection in swine with porcine respiratory disease complex.

We studied the occurrence of swine influenza virus (SIV) infection in piglets with respiratory symptoms resembling porcine respiratory disease complex (PRDC). A total of 106 samples including nasal swab and lung suspension from sick piglets were collected from 30 farms of medium size in the central and eastern parts of Thailand from August 2006 to February 2007. Samples were inoculated onto Mardin-Darby Canine Kidney (MDCK) cells and SIV infection was confirmed by immunofluorescent assay (IFA) and reverse transcriptase polymerase chain reaction (RT-PCR) specific for M gene. Of 106 samples, 3 pigs from 3 different farms were found to be SIV positive on all assays. The positive samples were further identified by RT-PCR as H3N2 subtype using specific primers for hemagglutinin (HA) and neuraminidase (NA) genes. SIV infection was found in 2.8% of swine suffering from respiratory distress suggesting SIV may not be the major pathogen for PRDC in the central and eastern Thailand. SIV was present in 3 of 30 farms (10%) indicating the prevalence of SIV in these regions is considerable. Since pigs are vulnerable to infection from both human and avian influenza viruses and interspecies transmission between humans and swine occurs sporadically, it is essential to continue surveillance and monitoring of SIV infection in the swine population.