Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3SL-PAA and 6SLN-PAA, which contained, respectively, 3-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q3L, increased binding to 6SLN-PAA, while among H1 swine viruses, the 190E3D and 225G3E mutations in the HA appeared important for the increased affinity of the viruses for 6SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

Genetic and biologic observations suggest that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.Influenza A viruses contain eight segments of negativesense, single-stranded RNA (reviewed in reference 16). Each RNA segment encodes at least one protein, and two of these proteins, hemagglutinin (HA) and neuraminidase (NA), project through the viral envelope and are available for interactions with cellular molecules. The abundance of each protein varies among virus subtypes, with the HA-NA ratio of influenza virus A/WSN/33 (H1N1) being approximately 10 to 1 (21). Since HA and NA recognize the same molecule (sialic acid) with conflicting activities, it can be assumed that drastic changes in either activity would affect viral replication.The HA, a type I integral membrane glycoprotein, is cleaved into two disulfide-linked chains, HA1 and HA2, by host proteases. Such cleavage is critical for viral infectivity, because it exposes the membrane fusion peptide located at the amino terminus of the HA2 subunit (reviewed in reference 14). The HA functions as a homotrimer of noncovalently linked monomers and plays two major roles during the replication of influenza A virus in host cells. First, it attaches the virus to the cell surface by binding to sialic-acid-containing receptors and promotes viral penetration by mediating fusion of the endosomal and viral membranes. The conserved sialic acid receptor-binding pocket, located on the HA1 subunit at the distal end of the molecule, binds to monovalent sialic acid receptor analogs with relatively low affinity (dissociation constant, approximately 0.1 to 1 mM [11]); however, the high abundance of HA molecules on the virion surface permits a sufficient number of low-affinity interactions to allow virus attachment and entry into host cells.The NA molecule, a type II integral membrane glycoprotein (7, 28), consists of a box-like catalytic head, a centrally attached stalk with a hydrophobic transmembrane-spanning region that attaches the molecule to the plasma ...

Background In February 2020, a locally-acquired COVID-19 case was detected in Lombardia, Italy. This was the first signal of ongoing transmission of SARS-CoV-2 in the country. The outbreak rapidly escalated to a national level epidemic, amid the WHO declaration of a pandemic. MethodsWe analysed data from the national case-based integrated surveillance system of all RT-PCR confirmed COVID-19 infections as of March 24 th 2020, collected from all Italian regions and autonomous provinces. Here we provide a descriptive epidemiological summary on the first 62,843 COVID-19 cases in Italy as well as estimates of the basic and net reproductive numbers by region.Findings Of the 62,843 cases of COVID-19 analysed, 71·6% were reported from three Regions (Lombardia, Veneto and Emilia-Romagna). All cases reported after February 20 th were locally acquired. Estimates of R0 varied between 2·5 (95%CI: 2·18-2·83) in Toscana and 3 (95%CI: 2·68-3·33) in Lazio, with epidemic doubling time of 3·2 days (95%CI: 2·3-5·2) and 2.9 days (95%CI: 2·2-4·3), respectively. The net reproduction number showed a decreasing trend starting around February 20-25, 2020 in Northern regions. Notably, 5,760 cases were reported among health care workers. Of the 5,541 reported COVID-19 associated deaths, 49% occurred in people aged 80 years or above with an overall crude CFR of 8·8%. Male sex and age were independent risk factors for COVID-19 death.Interpretation The COVID-19 infection in Italy emerged with a clustering onset similar to the one described in Wuhan, China and likewise showed worse outcomes in older males with comorbidities. Initial R0 at 2·96 in Lombardia, explains the high case-load and rapid geographical spread observed. Overall Rt in Italian regions is currently decreasing albeit with large diversities across the country, supporting the importance of combined non-pharmacological control measures.Funding: routine institutional funding was used to perform this work.

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